Guillaume Hatte

Claude Prigent

Jean-Pierre Tassan
email: jean-pierre.tassan@univ-rennes1.fr

Adherens junctions are involved in polarized contractile ring formation in dividing epithelial cells of Xenopus laevis embryos

Keywords: Polarised contractile ring formation apical basal adherens junctions cytokinesis, actomyosin, asymmetric furrowing, cell division, polarity

Adherens junctions are involved in polarized contractile ring formation in dividing epithelial cells of Xenopus laevis embryos

Introduction

Mitotic cells divide to produce two daughter cells in a process called cytokinesis, which relies upon contraction of an actomyosin ring placed at the cell equator. The contractile ring is constituted of actin filaments and the non-muscular motor protein myosin II [START_REF] D'avino | Cytokinesis in animal cells[END_REF]. In isolated cells, the actomyosin ring contracts symmetrically, ingressing the cell plasma membrane all around the mother cell midzone in an isoconcentric manner. The contractile actomyosin ring must be accurately positioned at the equatorial cortex to bisect the mitotic spindle and equally distribute the chromosomes. This appropriate positioning is ensured by signals emanating from the mitotic spindle in late anaphase. The building and contraction of the contractile actomyosin ring requires activation of the small regulatory GTPase RhoA by the GTPase Exchange Factor (GEF) Ect2. Centralspindlin, a heterotetramer of the kinesin-related protein MLKP1 and the GTPase-activating protein (GAP) MgcRacGAP, which interacts with microtubules of the central spindle, delivers Ect2 to the cell cortex, where it activates RhoA. Activated RhoA regulates simultaneously the polymerization of actin filaments (F-actin) and activation of non-muscular myosin II, thereby leading to constriction of the cytokinetic ring [START_REF] D'avino | Cytokinesis in animal cells[END_REF]. These regulations by RhoA happen via concomitant activation of formins which increases actin polymerisation and activation of the Rho-associated coiled-coil containing protein kinase II (Rock II). Rock II in turn phosphorylates myosin light chain, thus activating myosin. In addition to actin and myosin II, other proteins are components of the contractile ring, including the scaffolding protein anillin which anchors the contractile ring to the cell plasma membrane, maintains the furrow stability and is necessary for effective RhoA-mediated contractility [START_REF] Piekny | Anillin Is a Scaffold Protein That Links RhoA, Actin, and Myosin during Cytokinesis[END_REF][START_REF] Reyes | Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin[END_REF][START_REF] Straight | Anillin binds nonmuscle myosin II and regulates the contractile ring[END_REF][START_REF] Liu | Cleavage furrow organization requires PIP 2-mediated recruitment of anillin[END_REF].

In contrast to the isoconcentric furrowing that occurs when isolated cells divide, contraction of the actomyosin ring is polarized in cells dividing in the plane of an epithelium, likely reflecting their marked apical-basolateral polarity. Furrowing typically progresses from the cell's basal side towards the apical surface. This mode of cytokinesis by asymmetric furrowing has been reported in diverse model systems, including insects and vertebrates, suggesting that asymmetric furrowing is the default mode of cytokinesis in epithelia [START_REF] Bras | Epithelial cell division -Multiplying without losing touch[END_REF][START_REF] Tassan | Asymmetries in cell division, cell size, and furrowing in the xenopus laevis embryo[END_REF]. Asymmetric furrowing is intimately linked to epithelial cell polarity. Epithelial cells have distinct apical and basolateral plasma membrane domains, separated by a belt of tight and adherens junctions [START_REF] Gibson | Apicobasal polarization: Epithelial form and function[END_REF][START_REF] Rodriguez-Boulan | Organization and execution of the epithelial polarity programme[END_REF][START_REF] Tepass | The apical polarity protein network in drosophila epithelial cells: Regulation of polarity, junctions, morphogenesis, cell growth, and survival[END_REF][START_REF] Thompson | Cell polarity: Models and mechanisms from yeast, worms and flies[END_REF].

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Adherens junctions participate in the maintenance of epithelial tissue integrity. They are constituted of the transmembrane protein cadherin, which creates, via its extracellular domain, homophilic interactions with cadherin of neighbouring cells.

Through interactions of its intracytoplasmic tail with α and β -catenins, cadherin is indirectly connected to the apical actomyosin belt, which maintains epithelial cell shape and epithelial integrity.

In Drosophila, it has been shown that adherens junctions contribute to the asymmetry of furrowing of dividing cells [START_REF] Morais-De-Sá | Adherens junctions determine the apical position of the midbody during follicular epithelial cell division[END_REF][START_REF] Guillot | Adhesion Disengagement Uncouples Intrinsic and Extrinsic Forces to Drive Cytokinesis in Epithelial Tissues[END_REF]. The cytokinetic ring constriction, which is normally asymmetric in Drosophila epithelial cells, becomes symmetric in cells depleted of β-catenin or E-cadherin. These studies demonstrate the importance of adherens junctions for asymmetric furrowing and anchoring of the contractile ring for apical positioning of the midbody. However, it remains largely unknown how the contractile ring forms and when it becomes connected to adherens junctions.

Here, we used the epithelium of Xenopus laevis embryos at the gastrula stage to study contractile ring formation, because at this developmental stage, cells divide exclusively parallel to the epithelium plane such that both daughter cells remain continuously in that plane [START_REF] Chalmers | Oriented cell divisions asymmetrically segregate aPKC and generate cell fate diversity in the early Xenopus embryo[END_REF]. Moreover, we have previously shown that epithelial cells divide by asymmetric furrowing from the basal towards the apical side [START_REF] Page | A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development[END_REF].

Using live imaging of Xenopus laevis gastrula embryos expressing several contractile actomyosin ring components, we have here characterized the dynamics of contractile ring formation and have shown that the contractile ring formation is polarized from apical cell-cell junctions towards the cell basal side. Activation of the contractile motor protein myosin II leading to the contraction of the actomyosin ring is also polarized.

Finally, we shown that adherens junctions are involved in the formation kinetics of the contractile ring.

Materials and Methods

Plasmids

Plasmids pCS2-Lifeact-GFP (a kind gift from Ulrike Engel, Heidelberg University, Germany), pCS2-mRFP-GPI, pCS2-GFP-GPI (a kind gift from Mikael Brand,

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Dresden University of Technology, Germany), pCS2-GFP-rGBD (a kind gift from William Bement), pSp64T-C-cad Ctail [START_REF] Lee | Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin[END_REF], pT7T-ZO-1-GFP [START_REF] Hatte | Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue[END_REF] and pT7T-SF9-GFP [START_REF] Hatte | Epithelial cell division in the Xenopus laevis embryo during gastrulation[END_REF] were used in this study. pT7T-GFP-Rock II K/R was constructed by site-directed mutagenesis of lysine 117 (K117) from Xenopus Rock II (a kind gift from Johné Liu, [START_REF] Farah | A Rho-associated protein kinase, ROKα, binds insulin receptor substrate-1 and modulates insulin signaling[END_REF]). The K117, essential for the catalytic activity of the kinase, was replaced by an arginine to abolish Rock II activity because the overexpression of the active kinase causes division defects, which are not obtained with the Rock II K/R mutant. The amplification product was cloned into the vector pT7T in phase with N-terminal GFP.

PT7T-3xGFP-anillin was constructed by PCR amplification of the Xenopus anillin sequence, from the plasmid pT7T-GFP-Xlanillin [START_REF] Page | A functional analysis of MELK in cell division reveals a transition in the mode of cytokinesis during Xenopus development[END_REF] and subcloned into pT7T-3xGFP with N-terminal 3xGFP. All new constructs were verified by sequencing. All plasmids were linearised to generate mRNAs by in vitro transcription using the T7 or SP6 mMessage mMachine kit (Ambion).

α-catenin and ZO-1 knockdown

For α-catenin and ZO-1 knockdown experiments, antisense morpholinos targeting the initiation codon of respectively α-catenin (5′-ATGTTTCCTGTATTGAGAGTCATGC-3′) and ZO-1 (5′-GCCGGTGTCAGTATGAGTCCCCAGA-3′) were produced by Gene Tools. These morpholinos were previously characterized [START_REF] Hatte | Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue[END_REF][START_REF] Nandadasa | Regulation of classical cadherin membrane expression and F-actin assembly by alphacatenins, during Xenopus embryogenesis[END_REF]. A standard morpholino (5′-CTCTTACCTCAGTTACAATTTATA-3′, Gene Tools) was used as a control.

Preparation of Xenopus laevis embryos and microinjection

Xenopus laevis albinos were obtained from Biological Resources Centre (CRB, Rennes, France). All animal experiments were performed in accordance with the approved protocols and guidelines at Rennes 1 University by the Comité Rennais d'Ethique en Matière d'Expérimentation Animale (C2EA-07) and the French Ministry for Education and Research (3523813). Eggs were fertilized in vitro and embryos were collected as described previously [START_REF] Hatte | Epithelial cell division in the Xenopus laevis embryo during gastrulation[END_REF]. After de-jelling, embryos were placed in 5% Ficoll in F1 buffer (

Indirect immunofluorescence

The embryos were fixed and treated as previously described [START_REF] Hatte | Epithelial cell division in the Xenopus laevis embryo during gastrulation[END_REF].

Briefly, embryos were fixed with 2% trichloroacetic acid in 1× F1 buffer. After embryos were devitellinated, cells were permeabilised in PBS+1% Triton X-100 for 20 min at room temperature, incubated in PBS+0.1% Triton X-100 (PBST 0.1%) for 10 min and then blocked for 1 h in 2% BSA in PBST 0.1% (BSA-PBST). Embryos were incubated overnight in BSA-PBST at 14°C with a primary antibody.

The primary antibodies used in this study are directed against C-cadherin (DHSB, 6B6, 1:200), α-catenin (ThermoFisher Scientific, PA1-25081, 1:100), β-catenin (Santa-Cruz, SC-7199,1:100), F-actin (AC-40, Sigma, 1:200), phospho S20 myosin regulatory light chain (MRLC, pS19 in Xenopus laevis, abcam, ab2480, 1:100), MKLP1 (Santa Cruz, SC-867, 1:100), Ect2 (Millipore 1:100), Xenopus laevis anillin and myosin heavy chain A (MHCA) (a kind gift from Aaron Straight, [START_REF] Straight | Anillin binds nonmuscle myosin II and regulates the contractile ring[END_REF], 1:100), Xenopus laevis Rock II (a kind gift from Johné Liu, [START_REF] Farah | A Rho-associated protein kinase, ROKα, binds insulin receptor substrate-1 and modulates insulin signaling[END_REF] 1:100), ZO-1 (Zymed, 33-9100, 1:200), occludin (a kind gift from Sandra Citi [START_REF] Corderionsi | Occludin dephosphorylation in early development of Xenopus laevis[END_REF], 1:100), GFP (Roche, clone 7.1 and 13.1, 11914460001, 1:100) and mcherry (abcam, ab167453, 1:200).

After washing with PBST 0.1%, embryos were incubated with Alexa Fluor-488 or-555 anti-mouse, anti-rabbit or anti-sheep (ThermoFisher Scientific) and TO-PRO-3 to stain DNA (Invitrogen, 0.5 μg/ml) in BSA-PBST for 1 h at room temperature. After 3 washes of 5 min in 0.1% PBST, embryos were mounted in Vectashield (Vector).

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For F-actin staining, embryos were fixed in 3.7% formaldehyde in F1 buffer for 2 hours at room temperature, permeabilised and blocked as previously described. After blocking, embryos were incubated with AlexaFluor 555-conjugated Phalloidin (Life Technologies, 1 U/ml) and TO-PRO3 for 2 h, washed and mounted in Vectashield.

Lambda phosphatase treatment of embryos

Embryos fixed in TCA, as previously described, were incubated in the appropriate buffer with or without 25 units of lambda phosphatase (New England Biolabs) at 30°C for 30 min. Embryos were then extensively washed with PBS, permeabilized and proceed for indirect immunofluorescence with the anti-phospho S20 myosin regulatory light chain as the primary antibody.

Confocal microscopy on living and fixed embryos

For immunofluorescence, a Leica SP5 confocal microscope with an oil immersion objective 40x / 1.25-0.75 HC PL APO (WD 0.1 mm) was used. Imaging of live embryos was performed with a Leica SP8 confocal microscope with a 63x / 1.4 HC PL APO oil objective, using a resonant scanner. This scanner allows extremely fast acquisitions (scanning frequency of 8000 Hz), essential for analyzing the recruitment of proteins into the contractile ring.

Quantification of immunolabelling at adherens and tight junctions

To test if contractile ring activation could be polarized along the apical-basal cell axis, the fluorescence intensities of Rock II and pS19MRLC detected by immunolabelling in fixed embryos were quantified. Because Rock II localises at the apical junctional complex and is distributed along the lateral membrane several µm below (Fig. 1B), quantification of fluorescence intensity was standardized by measuring 10 µm below the apical cell-cell junctions. At this distance, the fluorescence intensity was minimal.

In contrast, pS19MRLC fluorescence is more restricted to apical cell-cell contacts compared to Rock II (Fig. 1B), therefore quantification was standardized by measuring 5 µm below apical junctions. For both Rock II and pS19MRLC, J o u r n a l P r e -p r o o f fluorescence intensities were also measured at non-furrowing faces, orthogonal to the division site (hereafter referred as cell poles). To quantify fluorescence intensity for Rock II and pS19MRLC, the mean pixel intensity was measured in a region of interest (ROI) at the cell-cell contacts. This ROI was placed at the division site for cells already engaged in cytokinesis or perpendicularly and equally distant from mitotic chromosomes. The mean fluorescence pixel intensity in proximity to the presumptive division site was measured in a ROI with identical size to the ROI used for quantification at the division site and was subtracted from the pixel intensity at the division site.

Quantification of the duration of ring contraction and contractile ring formation

The duration of the ring contraction was calculated by subtracting the time when the contractile ring begins to contract at the basal side to the time when it is fully ingressed and concentrates at the apical pole of the cell. For the duration of the contractile ring formation, the time when the protein of interest begins to accumulate at the presumptive division site was subtracted from the time when the contractile ring begins to constrict at the basal side.

Image processing and quantification

Images were processed using ImageJ software (Rasband, W.S., http://rsb.info.nih.gov/ij) and figures were prepared using ImageJ and Photoshop.

Graphing was performed using Microsoft Excel. Kymographs of lateral cell-cell contacts were mounted manually.

Statistical analysis

No statistical method was used to predetermine the sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment. Experiments were repeated at least two times. Sample sizes are indicated for each experiment in figure legends. A two-tailed unpaired Student t-test was applied to determine statistical significance. Statistical analysis was performed using Microsoft Excel software except for Rock II and J o u r n a l P r e -p r o o f pS19MRLC fluorescence pixel intensities at the division sites and poles for which a Mann Whitney test was done using Prism software.

Results

The proteins involved in cytokinesis localize at the apical junctional complex in the Xenopus laevis embryo

First and foremost, we studied the formation of the actomyosin contractile ring by examining the localization of several proteins involved in cytokinesis in the epithelium of Xenopus laevis embryo. In non-dividing cells, filamentous actin (F-actin) and myosin heavy chain A (MHCA) localize at apical cell-cell contacts where they form a contractile belt at the apical cell periphery (Fig. 1A). On orthogonal projections, proteins appear as dots extended by faint signals which intensity decreases along the lateral cell-cell contacts (Fig. 1A, arrowheads on orthogonal views). The F-actin signal decreases along the lateral cell cortex whereas β-catenin was localized all around the basolateral cell contacts (Fig. 1B; [START_REF] Woolner | Spindle Position in Symmetric Cell Divisions during Epiboly Is Controlled by Opposing and Dynamic Apicobasal Forces[END_REF]) indicating that the decrease of Factin signal is specific. MHCA was also detected in the cytoplasm and at the medial cortex. Anillin, a scaffolding protein which interacts with F-actin and myosin, also localise at the apical cell-cell contacts and into nuclei (Fig. 1A), as described previously [START_REF] Reyes | Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin[END_REF]. As previously shown in cultured cells [START_REF] Ratheesh | Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens[END_REF], the two regulators of cytokinesis, MKLP1, a constituent of the centralspindlin complex and Ect2, an activator of Rho GTPase, localize at apical cell-cell contacts (Fig. S1A andS1C). We also examined the localisation of the protein kinase Rock II which activates myosin by phosphorylating the serine 20 residue (serine 19 in Xenopus laevis) of the myosin regulatory light chain (MRLC) leading to the contraction of the actomyosin contractile ring during cytokinesis [START_REF] Amano | Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase)[END_REF]. The activated MRLC was detected with an antibody recognizing the MRLC serine 19 phosphorylation (pS19MRLC). Both proteins, Rock II and activated myosin, co-localize with C-cadherin at the adherens junctions, although pS19MRLC was more restricted towards apical side unlike Rock II (Fig. 1C).

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Our results show that all the key proteins involved in cytokinesis that we examined localize to the apical cell-cell contacts in the Xenopus embryo epithelium.

Accumulation of contractile ring components begins at apical cell-cell contacts and expands basally along the lateral membrane

To study the formation dynamics of the actomyosin contractile ring in epithelial cells, we expressed four distinct fluorescent proteins in embryos. We expressed the two proteins involved in cytokinesis, Rock II K/R, a catalytically inactive form of Rock II and anillin tagged with GFP (respectively GFP-Rock II K/R and 3xGFP-anillin) and used two fluorescent probes: the SF9-GFP intrabody, which detects the endogenous myosin heavy chain A (MHCA) [START_REF] Nizak | Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo[END_REF], and GFP-Lifeact which detects F-actin [START_REF] Riedl | Lifeact: A versatile marker to visualize F-actin[END_REF]. As described above for endogenous proteins, all the four proteins localized at the apical cell-cell junctions (Fig. 2 A-D and Fig. S2A). Expression of these fluorescent proteins has no significant impact on the duration of ring contraction compared with cells expressing GFP fused with a glycosylphosphatidylinositol (GPI)-anchor (GFP-GPI) used to visualize cells plasma membrane (Fig. S2B, mean duration of ring contraction 5 min 50 s). During cytokinesis, the four proteins that we tested localize at the contractile ring (Fig. 2A-D, white arrowheads on front views, see Fig. S2C for an example of full cytokinesis and asymmetric constriction of the contractile ring in a cell expressing 3xGFP-anillin). Interestingly, very shortly before plasma membrane ingression starts at the basal side, the fluorescence intensity of the four proteins increases at the apical cell-cell contacts at the presumptive plasma membrane ingression site (Fig. 2 A-D, dashed circles on front views, orthogonal views). The extent to which fluorescence intensities increased depended on the fluorescent protein, with 3xGFP-anillin increasing more than the 3 others (Fig. 2 A-D

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In epithelial cells of the Xenopus embryos, the mitotic spindle position along the apicobasal axis is stereotypically biased toward the apical surface (Fig. S4 and [START_REF] Woolner | Spindle Position in Symmetric Cell Divisions during Epiboly Is Controlled by Opposing and Dynamic Apicobasal Forces[END_REF]).

Because in late anaphase, the mitotic spindle signals to the cell cortex the position of the contractile ring, the asymmetrically positioned mitotic spindle could be involved in the apical-to-basal accumulation of contractile ring proteins. Interestingly, the two cytokinesis regulators, MKLP1 and Ect2, localized at apical and lateral cell-cell contacts in anaphase cells (Fig. S1B and S1D, respectively). This result suggests that the asymmetrically positioned mitotic spindle could drive the apical recruitment of the contractile ring proteins.

In summary, the four proteins involved in cytokinesis, which we examined, start to accumulate at the presumptive division site asymmetrically from the apical cell-cell contacts and expand along the lateral cell plasma membrane towards the basal side.

Activation of the actomyosin contractile ring is polarized

The finding that contractile ring components accumulate asymmetrically led to the question of whether activation of the actomyosin contractile ring could be polarized along the apical-basal cell axis. Since RhoA is a master regulator of actomyosin ring activation, we thought to use the GFP-rGBD probe [START_REF] Benink | Concentric zones of active RhoA and Cdc42 around single cell wounds[END_REF] to follow active RhoA in living embryos. Fluorescence of the GFP-rGBD probe was detected at cell-cell contacts in non-dividing cells and at the contractile ring in cells advanced in cytokinesis (Fig. S5A). However, the fluorescence in the cytoplasm was also high (cells expressing high and low levels of GFP-rGBD are shown in Fig. S5A) which turned out to be incompatible with the high temporal resolution needed to image GFP-rGBD at the presumptive division site. This issue made the GFP-rGBD probe unusable for our study on the early steps of cytokinesis. To overcome this challenge, we investigated the localization of the myosin II regulatory light chain phosphorylated on serine 19 (pS19MRLC) in fixed embryos. S19 phosphorylation, which is catalysed by Rock II, induces myosin activation and subsequently actomyosin constriction. To validate the approach based on fixed material, we first studied Rock II localization, as we have previously shown the polarized accumulation of GFP-Rock II KR at the presumptive division site. Immunofluorescence signal of Rock II was indistinguishable between cells in metaphase and at the very beginning of anaphase, when chromosomes start J o u r n a l P r e -p r o o f splitting (these cells will be referred as early anaphase, Fig. 3A). However, in cells more advanced in anaphase, Rock II signal was detected deeper along the lateral plasma membrane. Hereafter, these cells advanced in anaphase, showing Rock II accumulation but without any sign of plasma membrane ingression will be referred as early cytokinesis (Fig. 3A, compare orthogonal views at the division site of early anaphase with early cytokinesis). In cells more advanced in cytokinesis, the ingressed plasma membrane was generally fully labelled by the anti-Rock II antibody (Fig. 3A). However, for some unknown reason, we also frequently observed incomplete labelling at the base of the contractile ring. A similar observation of incomplete ring labelling was previously reported in living tissues of Drosophila [START_REF] Founounou | Septins Regulate the Contractility of the Actomyosin Ring to Enable Adherens Junction Remodeling during Cytokinesis of Epithelial Cells[END_REF].

Rock II fluorescence intensity was quantified at the presumptive division site in early anaphase and early cytokinesis cells. For comparison, in the same cells, we also quantified Rock II signal at the cell poles (non-furrowing faces orthogonal to the division site). The quantitative analysis shows a significant statistical difference between Rock II intensity at the presumptive division site compared to cell poles (Fig. 3B). Moreover, at the presumptive division site, Rock II signal intensity increases with the length of plasma membrane labelled for Rock II (Fig. 3C). Although Rock II signal intensity also increases at the cell poles, this increase was less marked compared to the presumptive division site (Fig. 3C, equation of linear regressions are given for poles and division site). The correlation between Rock II accumulation and the length of the plasma membrane labelled for Rock II suggests that the Rock II signal expands along the baso-lateral membrane. This result is in agreement with the previously shown polarized accumulation of the GFP-Rock II K/R probe in living embryos. Therefore, we used indirect immunofluorescence with fixed embryos to study myosin activation.

Fixed embryos were labelled with an anti-pS19 MRLC antibody, the specificity of which was previously validated (Fig. S5B). As previously observed for Rock II, pS19MRLC signal was indistinguishable between cells in metaphase and early anaphase (Fig. 3D). As previously quantified for Rock II, fluorescence intensities were also quantified at the division site and at the cell poles. Quantifications show a significant statistical difference between pS19MRLC fluorescence intensity at the presumptive division site compared to cell poles (Fig. 3E). Moreover, pS19MRLC signal intensity increases with the length of the plasma membrane labelled for J o u r n a l P r e -p r o o f pS19MRLC at the presumptive cell division site whereas it remained almost unchanged at the cell poles (Fig. 3F). These results are consistent with the phosphorylation of the myosin regulatory light chain being polarized, starting at the apical cell-cell junctions and spreading along the lateral membrane towards the basal side.

Altogether, our results show that accumulation of proteins constituting the actomyosin contractile ring, and activation of the ring start at the apical cell-cell contacts and expands along the lateral membrane. Since furrowing progresses in the basal to apical orientation, the reverse polarity of the contractile ring formation was unexpected.

The adherens junctions are involved in the polarized formation of the contractile ring

Our results show that cytokinetic proteins co-localise with adherens junctions and the assembly and activation of the cytokinetic contractile ring begins at the apical cell-cell contacts. Due to the close link previously shown between cytokinesis and the adherens junctions [START_REF] Morais-De-Sá | Adherens junctions determine the apical position of the midbody during follicular epithelial cell division[END_REF][START_REF] Guillot | Adhesion Disengagement Uncouples Intrinsic and Extrinsic Forces to Drive Cytokinesis in Epithelial Tissues[END_REF][START_REF] Benink | Concentric zones of active RhoA and Cdc42 around single cell wounds[END_REF][START_REF] Founounou | Septins Regulate the Contractility of the Actomyosin Ring to Enable Adherens Junction Remodeling during Cytokinesis of Epithelial Cells[END_REF][START_REF] Higashi | Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis[END_REF], we examined if adherens junctions could be involved in the asymmetric formation of the contractile ring.

To interfere with the adherens junctions, we followed two distinct approaches. In the first approach, we used a deletion mutant corresponding to the cytosolic domain of Ccadherin (Ctail) [START_REF] Lee | Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin[END_REF]. C-cadherin (also termed EP-cadherin) is the main adherens junctions molecule of the Xenopus gastrula [START_REF] Choi | A cadherin-like protein in eggs J o u r n a l P r e -p r o o f and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone[END_REF][START_REF] Ginsberg | Expression of a novel cadherin (EPcadherin) in unfertilized eggs and early Xenopus embryos[END_REF]. Because the extra-cellular and the trans-membrane domains are missing in the Ctail, it is unable to integrate the plasma membrane and bridge with C-cadherin of neighbouring cells. However, the cytosolic domain of E-cadherin is responsible for indirect binding with the F-actin via interaction with α and β catenins. It has been shown that the ubiquitous expression in the Xenopus embryos of high levels of Ctail provokes cell dissociation [START_REF] Lee | Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin[END_REF]. This loss of cell-cell contacts indicated a decrease in cell adhesion suggesting that the Ctail could act as a dominant-negative mutant by titrating catenins. Because the mode of action of Ctail was not described in details, we further characterized this mutant. The Ctail mutant was co-expressed together with GFP or RFP -GPI used as tracers, and we examined the expression of diverse proteins localized at the adherens junctions.

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Unlike control cells, the apical membrane of cells expressing Ctail was frequently round (Fig. 4A, compare orthogonal views of control and Ctail), suggesting a decrease in cell-cell adhesion as previously reported by Lee and Gumbiner [START_REF] Lee | Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin[END_REF]. The amounts of endogenous C-cadherin, α and β-catenins, F-actin and Rock II, localized at the adherens junctions, were significantly decreased in cells expressing Ctail (Fig. 4A,histograms). In contrast, localization of two constituents of the tight junctions, ZO-1 and occludin, was not affected (Fig. S6). Our results show that expression of Ctail disrupts the adherens junctions, which could explain epithelial structural defects observed in our and previous studies [START_REF] Lee | Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin[END_REF].

We then tested if the expression of the Ctail mutant could affect the formation and contraction of the actomyosin ring. In both control and Ctail expressing cells, Lifeact-GFP and 3xGFP-anillin fluorescence appears apically and expands along the lateral membrane (Fig. 4B and4D, upper rows). The asymmetric contraction of the actomyosin ring from the basal side towards the apical side was also unaffected by Ctail expression (Fig. 4B and4D, lower rows).

Interestingly, the time elapsed between the beginning of the accumulation of Lifeact-GFP and 3xGFP-Anillin at the apical cell-cell contacts and the beginning of plasma membrane ingression at the basal side significantly increased in Ctail expressing cells compared to controls (58.9+/-7.2 s vs 30.7+/-10.4 s for Lifeact-GFP and 54.2+/-13.1 s vs 21.4+/-5.9 s for 3xGFP-Anillin, respectively Fig. 4C and4E). This result suggested that the adherens junctions could be involved in the polarized recruitment of the contractile ring components. To test this hypothesis, we followed a second approach to interfere with the adherens junctions.

In the second approach, we used an antisense morpholino oligonucleotide to deplete α-catenin, which indirectly links C-cadherin to the actomyosin cytoskeleton (Mo α-cat, Fig. 5A). As previously shown [START_REF] Nandadasa | Regulation of classical cadherin membrane expression and F-actin assembly by alphacatenins, during Xenopus embryogenesis[END_REF][START_REF] Hatte | Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue[END_REF], depletion of α-catenin also led to a significant decrease of C-cadherin (Fig. 5A) and perijunctional actin filaments (not shown, [START_REF] Hatte | Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue[END_REF]).

This result shows that Mo α-cat, by depleting α-cat, destabilizes the adherens junctions. Measurement of the time lasting from the beginning of fluorescence increase and the start of ring constriction shows that the recruitment durations of Lifeact-GFP and 3xGFP-anillin at the apical cell-cell contacts significantly increases in Mo α-cat treated cells compared with control morpholino (MoCo) cells (57.3+/-13.0 s vs 33.1+/-11.0 s for Lifeact-GFP and 41.5+/-8.0 s vs 27.6+/-4.0 s, respectively Fig.

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5B and 5C, histograms). As with the Ctail mutant, the polarized recruitment of Lifeact-GFP and 3xGFP-anillin occurred in cells treated with MoCo as well as cells treated with Mo α-cat (Fig. 5B and5C). The subsequent asymmetric ring constriction also normally occurred (not shown and [START_REF] Hatte | Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue[END_REF] for Lifeact-GFP). To test if tight junctions may contribute in maintaining the asymmetric ring constriction in α-cat depleted cells, embryos were microinjected with Mo α-cat, Mo ZO-1 and 3xGFP-anillin mRNA. ZO-1 is a tight junction-associated protein. As previously shown with ZO-1 disruption alone [START_REF] Hatte | Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue[END_REF], disruption of tight and adherens junctions led to prolonged cytokinesis and flattened contractile ring with normal asymmetry (Fig. S7).

Altogether, these results indicate that the adherens junctions are involved in the polarized accumulation of the contractile ring components at the presumptive division site.

Discussion

In the present study, we have shown that at cytokinesis onset the levels of proteins forming and activating the contractile actomyosin ring increase at the apical cell-cell contacts. The initiation of ring formation at the apical contacts could appear in contradiction with the asymmetric furrowing which progresses from the basal side towards the apical side. However, our results show that the ring formation is extremely rapid, occurring in approximatively 30 seconds before the first sign of ring constriction. Moreover, we have shown that ring formation spread laterally from apical contacts towards the basal side of cells.

Cytokinesis by asymmetric furrow ingression is closely linked to the presence of an apical junctional complex in epithelial cells. Indeed, the actomyosin contractile ring remains associated with apically localized adherens junctions in Drosophila [START_REF] Morais-De-Sá | Adherens junctions determine the apical position of the midbody during follicular epithelial cell division[END_REF][START_REF] Guillot | Adhesion Disengagement Uncouples Intrinsic and Extrinsic Forces to Drive Cytokinesis in Epithelial Tissues[END_REF].

In vertebrates, the apical junctional complex continuously contacts the contractile ring [START_REF] Baker | Epithelial cells retain junctions during mitosis[END_REF][START_REF] Higashi | Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis[END_REF][START_REF] Jinguji | Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine[END_REF]. Moreover, in Drosophila, it has been shown that interfering with the adherens junctions causes the furrow to become symmetric [START_REF] Morais-De-Sá | Adherens junctions determine the apical position of the midbody during follicular epithelial cell division[END_REF][START_REF] Guillot | Adhesion Disengagement Uncouples Intrinsic and Extrinsic Forces to Drive Cytokinesis in Epithelial Tissues[END_REF]. Although these observations showed that the contractile ring is associated with the adherens junctions, they did not elucidate when the two structures become associated. In the J o u r n a l P r e -p r o o f present study, we show that the adherens junctions are involved in the polarized contractile ring formation.

Although interfering with adherens junctions increased the duration of accumulation of proteins forming the contractile ring, it did not abolish the asymmetric contraction of the ring. These results are in contrast with previous studies in Drosophila in which it was shown that knockdown by RNAi of E-cadherin and α-catenin or in armadillo (the Drosophila orthologue of β-catenin) mutant, constriction of the actomyosin ring becomes symmetric [START_REF] Morais-De-Sá | Adherens junctions determine the apical position of the midbody during follicular epithelial cell division[END_REF][START_REF] Guillot | Adhesion Disengagement Uncouples Intrinsic and Extrinsic Forces to Drive Cytokinesis in Epithelial Tissues[END_REF]. The remaining amounts of junctional proteins at the adherens junctions observed in Ctail expressing cells and α-catenin knocked down cells could be sufficient to maintain asymmetric constriction of the contractile ring as noticed in our study. However, in Ctail cells, we frequently observed a rounding of cell indicative of a decrease in cell adhesion, which should have affected the asymmetric furrowing. The discrepancy between our results and results obtained in Drosophila regarding the asymmetric ring constriction may originate from the distinct models used. The organization of adherens and tight (septate in Drosophila) junctions differ in vertebrates and insects. Moreover, cytokinesis also exhibit marked difference in the two models including the maintenance of contacts of apical junctions with the contractile ring [START_REF] Higashi | Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis[END_REF]. It is possible that the tight junctions, by maintaining mechanical strain on the cell plasma membrane, could be involved in the maintenance of asymmetric constriction of the contractile ring. In this respect, we observed that tight junctions were unaffected by expression of the Ctail mutant. The double disruption of tight and adherens junctions led to flattened contractile ring and altered cytokinesis duration (Fig. S7), as previously shown for tight junction disruption alone which increases mechanical tension applied on adherens junctions [START_REF] Hatte | Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue[END_REF]. In the double knockdown, it is possible that ZO-1 depletion could lead, via remaining adherens junction proteins, to an increase of mechanical tension applied on adherens junctions and normal ring contraction asymmetry. As the mechanism leading to the increase of mechanical tension applied to adherens junctions upon ZO-1 depletion is unknown, the hypothesis of tight junctions involvement in asymmetric contraction of the contractile ring will require further investigations.

In epithelial cells of Xenopus embryos, the mitotic spindle position is stereotypically biased toward the apical cell side [START_REF] Woolner | Spindle Position in Symmetric Cell Divisions during Epiboly Is Controlled by Opposing and Dynamic Apicobasal Forces[END_REF] and (Fig. S4). This result suggests that the asymmetric apicobasal position of the mitotic spindle could be involved in J o u r n a l P r e -p r o o f accumulation of contractile ring proteins starting at apical cell-cell contacts of the presumptive division site. Our results suggest that in dividing epithelial cells, the signalling pathway leading to the contractile ring activation could be focused on adherens junctions (Fig. 5D). As microtubules are known to be involved in the signalling which induces cytokinesis [START_REF] D'avino | Cytokinesis in animal cells[END_REF], it will be of interest to explore their putative implication in the polarized accumulation of contractile ring proteins.

Interestingly, all proteins involved in cytokinesis analysed in the present study localize at the apical cell-cell contacts before cytokinesis onset. The previous studies showing that the centralspindlin complex, Ect2 and anillin regulate the Rho GTPase cycle at the adherens junctions indicated that they could participate in apical junctions' homeostasis [START_REF] Reyes | Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin[END_REF][START_REF] Ratheesh | Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens[END_REF][START_REF] Rosa | Ect2/Pbl Acts via Rho and Polarity Proteins to Direct the Assembly of an Isotropic Actomyosin Cortex upon Mitotic Entry[END_REF]. Conversely, it has been shown that depletion of αcatenin in MCF-7 epithelial cells reduces Ect2 levels at the adherens junctions [START_REF] Ratheesh | Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens[END_REF],

indicating that the adherens junctions are involved in recruiting components involved in the actomyosin contractility of the perijunctional belt in non-dividing cells.

Consequently, our results showing that adherens junctions are involved in the ring formation extend their implication to the contractile ring components during cytokinesis. Additional studies will be required to understand in details the highly dynamic mechanisms supporting the recruitment of cytokinetic proteins at the onset of epithelial cell cytokinesis.

Studies of cytokinesis in epithelial tissues have revealed the basal to the apical asymmetry of the contractile ring contraction. However, before contraction, the cell plasma membrane and the underneath contractile ring need to be freed from extracellular contacts at their basal side [START_REF] Bourdages | Quantitative analysis of cytokinesis In Situduring C. Elegans postembryonic development[END_REF][START_REF] Fishkind | Orientation and three-dimensional organization of actin filaments in dividing cultured cells[END_REF][START_REF] Taneja | Focal adhesions control cleavage furrow shape and spindle tilt during mitosis[END_REF]. Our study raises the question of how epithelial cells precisely orchestrate polarized contractile ring formation and activation with basal membrane disengagement.

Conclusions

Our study highlight the involvement of adherens junctions in the polarized formation and activation of the actomyosin contractile ring at the onset of cytokinesis. Our results give a simple explanation to the previously reported association of the actomyosin contractile ring with apical junctional complex during cytokinesis.

J o u r n a l P r e -p r o o f

  10 mM HEPES pH 7.6, 31.2 mM NaCl, 1.75 mM KCl, 59 μM MgCl2, 2 mM NaHCO3, and 0.25 mM CaCl2) for microinjection. mRNAs were J o u r n a l P r e -p r o o f microinjected into one blastomere of 4-or 8-cell-stage embryos. For α-catenin knockdown experiments, 20 ng of α-catenin antisense morpholino in 9.2 nl was microinjected into one blastomere at the 8-cell stage. For α-catenin and ZO-1 double knockdown, respectively 20 ng and 40 ng of morphilonos were microinjected. Embryos were then placed at 16°C overnight and analyzed at the gastrula stage (stage 11; [21]).

  , histograms). Subsequently, fluorescence spreads and fluorescence intensity increases with time along the baso-lateral membrane (Fig. 2 A-D, kymographs and histograms). The time elapsed between the beginning of accumulation of the four proteins at the apical cell-cell contacts and the beginning of plasma membrane ingression at the basal side ranges from 12 s. to 55 s. (Fig 2E, mean time: 30 seconds). Such a dynamic localization does not occur for GFP-GPI nor for the tight junction protein ZO-1, which follows the plasma membrane shape modifications without additional accumulation compared to non-dividing cells (Fig. S3A and S3B).

Fig. 1 .

 1 Fig. 1. Proteins involved in cytokinesis localize at the apical junctional complex. (A) F-actin was detected with fluorescent phalloidin and myosin heavy chain A (MHCA) and anillin by indirect immunofluorescence with specific antibodies. DNA stained with TO-PRO-3 is shown in inset. Orthogonal projections along the dashed lines are shown below apical views. Arrowheads point to the concentration of the labelling at the apical and lateral junctions. (B) Orthogonal projections of cells labelled by indirect immunofluorescence of F-actin and β-catenin. DNA stained with TO-PRO-3 (grey) was merged with F-actin and β-catenin. Arrowheads point to the concentration of the labelling at the apical and lateral junctions. (C) Co-labelling by indirect immunofluorescence of Rock II and phosphorylated S19MRLC (pS19MRLC, activated myosin) with the adherens junctional protein C-cadherin. The confocal planes passing through the apical junctions and overlays are shown. Orthogonal projections along the dashed lines are shown. Arrowheads point to the concentration of the labelling at the apical junctions. Scale bars: 10 µm.

Fig. 2 .

 2 Fig. 2. Accumulation of cytokinetic proteins begins at the apical junctions and further spreads basally along the lateral plasma membrane. Lifeact-GFP (A), SF9-GFP (B), 3xGFP-anillin (C) and GFP-Rock II KR (D) were expressed in living embryos and imaged. For each fluorescent protein, the representative still images of a dividing cell are shown. Time 0 corresponds to the start of fluorescent protein accumulation at the apical cell-cell contacts (time is indicated in seconds). Black arrowheads indicate ring contraction onset at the basal side. White arrowheads points on the accumulation of fluorescent proteins at the contractile ring. Below: orthogonal projections of a single lateral cell-cell contact (indicated by a dotted circle in front views) from the onset of fluorescent protein accumulation to the onset of ring contraction. Histograms show relative fluorescence intensity vs cell-cell contacts length at the onset of fluorescent protein accumulation (blue) and at the onset of ring contraction (red). a. : apical, b. : basal. ***: p-values < 0.001. Scale bars: 10 µm. (E) Duration of cytokinetic proteins accumulation at the division site. The time between the onset of proteins accumulation and actomyosin

Fig. 3 .

 3 Fig. 3. Activation of the actomyosin contractile ring is polarized. Indirect immunofluorescence for Rock II (A) and pS19MRLC (D). Surface views (Z0) and confocal planes 10 and 5 µm below are shown for respectively Rock II and pS19MRLC. Inset shows DNA detected with TO-PRO-3. Cells in metaphase, early anaphase, cytokinesis onset and advanced cytokinesis are presented for each antibody used. Orthogonal projection along the dashed lines are shown below. The horizontal and vertical dashed lines (shown only for metaphase cells) indicate the plane used to obtain orthogonal projection, respectively at the division sites and at the poles. Histograms B and E show pixel intensity of respectively Rock II and pS19MRLC fluorescence at the division site (blue circles) and poles (orange circles). The positions where the fluorescence intensity was measured are marked by blue (division site) and orange (poles) arrowheads on orthogonal projections. n= 50 for Rock II and 45 for pS19MRLC. Histograms C and F show pixel intensity of respectively Rock II and pS19MRLC fluorescence at the division site (blue circles) and poles (orange circles) as a function of the length of the lateral cell-cell contact labelled for respectively Rock II and pS19MRLC. Equations of the linear regressions are specified in the top right corner. Scale bars:10 µm.

Fig. 4 .

 4 Fig. 4. Destabilization of the adherens junctions increases the duration of cytokinetic proteins accumulation but does not impact the polarity of cytokinetic proteins recruitment and asymmetric ring constriction. (A) Expression of the C-cadherin deletion mutant, Ctail, destabilizes adherens junctions. Embryos expressing GFP-gpi or RFP-gpi alone (control) and co-expressing Ctail with GFP-gpi or RFP-gpi used as tracers were fixed and processed for indirect immunofluorescence with antibodies against C-cadherin, α and β-catenins and Rock II or with fluorescent phalloidin (to detect F-actin). GFP-gpi and RFP-gpi tracers were detected by immunofluorescence with antibodies against GFP and RFP, respectively. Apical confocal planes and orthogonal projections are shown. DNA is shown on

Fig. 5 .

 5 Fig. 5. Depletion of α catenin increases the duration of ring formation. (A) Endogenous α catenin and C-cadherin in embryos injected with α-catenin morpholino and RFP-membrane mRNA used as a tracer. For each condition, an image of a field containing cells expressing and not expressing RFP-membrane (asterisks) is shown. Red and blue bars on merged images indicate cell-cell contacts where fluorescence was quantified. Cells that do not express RFP-membrane are used as an internal control for fluorescence quantification. Line scans on the right show α-catenin and C-cadherin pixel intensity in internal control (blue) and cells expressing the RFP-membrane. Scale bars: 10 µm. (B) Kymograph of a baso-lateral plasma membrane at the site of division of a cell expressing Lifeact-GFP in control or α catenin morpholino. Histogram is the time of Lifeact-GFP accumulation in control or α-catenin morpholino (n=12 and 24 for respectively control and α catenin morpholinos). The p-value is indicated. (C) Same as in B with 3xGFP-Anillin instead of Lifeact-GFP. (n=9 and 19 for respectively control and α catenin morpholinos) (D) Schematic representation of cytokinesis initiation in polarized epithelial cells. The mitotic furrow, which is asymmetrically positioned along the apicobasal axis, signals

Fig. S7 .•

 S7 Fig. S7. (related to Fig. 5): Cells expressing 3xGFP-anillin in control or α-catenin and ZO-1 morpholinos. For each condition, still images of a representative dividing cell are shown. Apical views are shown. Below, orthogonal projections through the plan parallel to the division site indicated by a dotted line. Times are indicated in seconds. Scale bars: 10 µm.

Acknowledgements

We are grateful to Michael Danilchik, Arun Prasath Damodaran (C. Prigent's team) and Roland Le Borgne for critical reading on the manuscript. We thank the Microscopy Rennes Imaging Center (MRic, BIOSIT, IBiSA). This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Ligue Contre le Cancer (comités 29 et 35). G.H. was supported by the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation and partly by a grant from the Ligue Nationale Contre le Cancer. 2 J o u r n a l P r e -p r o o f

Author contributions

G.H. carried out the experiments, generated and interpreted the data. J-P.T. designed research and supervised the project. G.H. C.P. and J-P. T. wrote the manuscript.

Conflict of interest

The authors declare no conflict of interest.

Supplementary figure legends

J o u r n a l P r e -p r o o f

Conflict of interest

The authors declare no conflict of interest.

J o u r n a l P r e -p r o o f


The Effect of an Axial Force on the Vibration of Hinged Bars BY S .

WOJNOWSKY•KRIEGER1

It can be shown that the vibration of an exten&ihle bu, can:ying no tmnaverse load and havh:tg the ends fixed at the l!lupports, causes an axial tensile force with a period equal to the half�period qf the vibration of the bat, This m� modifies the process. of th'.:! vibration to a -nonlinear one and produ� an increase of th.e frequency ofvibrati(ln a«:ording to the Wc.-ease of the amplitude.

NOMENCU TUllE

The follnwing nomenclature is used in the po.per: llength of bar y = inst.Antaneous deflection of a.ny point dimentdons; however, it must be small enough to t"epreisent th1:1 curvature of the defiooted J?u: by the approximate exprossi on b"i;/b.w'.

In the absence of txa.ns veroo load, the deftection of the vibra.ting bar, Fig .

l, ia defined by the differential equo.tion

O• y b y� O'tJ B �• "" -µ._ b t• +(So+ 81) �•,,, •..... {lJ , ra. l
The value So being gi�, we will first ex}lreSfl the unknown force 81 through the dellootion y. The amount of &pproaoh of both hinged ends of the bar due to the deJleetion is

Al -�f,1 (�) ' @r •••••••••••• •••• [2]
Now, the axial force 81 produ!lel! "'°elongation of the bar

(I t) S,l ( EA) Al =S 1 EA-(i "'EA 1-li , ....... [ 3 ]
It may be seen from the last e.xprossi on that the constant 1/fJ l.'itL1l be omitted in our further investiption without any loae of geuerality. lf, in fact, the actual 1101lll l tant 1 /If

•••••••• : •• {5)
Substituting the last expxeMion in Equation.

[1]1 we. obtain The effect of the amplitudeon the incrense ot the rreq�ncy b.

!lOO n from Table 1.

.. • • • ••• • [24)
. where 'i' dllJloteM a quadric in '/I. The invenie of this integral is again a.n elliptic function. Contrary W the result given in Equa tion [13J, it would represent a no nsym.m.etr:imU n'bratiori abo1tt. the axi.811 "'" 0 of the undA!fleeted bar. Soi;.UTION BT B£SSE L FtrNCTION:!I A. solu.tion analogous to Eq_ua.tion [14] can be obtained. b/ J meMS of Be:mel functi� for the ease of a vibrating" circular plaOO wiUl fixed edgq:, if tml defioot.ed surfa.ee of the plate-ia a&rumed a surface-of rovolution.. B'.owever, sueh a solution would be of little prn.etical interest. In fact, if the amplitude of r. he vibt11-tion of the plate remains sina.11, es eompMOd with its t.hicknem, the i.nfluence of the e�rnal forces a.etingin the middle plane can be practjeaJly :D.eglected. However, if the defteutio ns of the plate p.re Cl�l'tlparable with its thi� the well-known theory of bending of pla.tes with large de:fleotionfi must be taken $8 the basis tor further investigation, and th& whole problem of vibrations then becomea much JilOl'9 complicated.

•

  '11 � __ B_ O'!/ + ! [s, + _!l_f.(°')' ,,.]•'11 ..

.

  •-.;s1n-, -...... . ... . ... ... !71 the ge(nlll.'l trical eoriditfurui at the hinged ends of the bar are

  -;; •....••.•••...•..•.•• [161 In the absence of axial forees (we can get this e98e � s� = O, r .... . "" and thus obtaining a "" O, k ,.. O, K = r/2) the frequency beoome!I «!G = Pf! .•..•..........••.... (171where po• ""' n•T•B/,d•. Coropa.ring this weJJ,.known resnlt with the caoo in which the ends of the bar a.re fixed {S, � 0), but the initial axial force ia zero (S� = O}, we have by Equa tion[IOI ...... [18)

  critiCll.l value of the compreooi ve fortle in a vi bra. ting bar Di lar;er than the Euler load P-.. -when the deflection i;; zero, and it decrell.ses to the. wlueP = P�-l')'.Ul.X S1 = P�•••••••••• ...1221 at the instant of the rna.ximum detleetion (,P ... 1). Until now, n.o tr�verse load on the bar bas been assumed . T:too ting the I)tQblem more rigorouely, we should introduce at leMt a load eommem>urate to the mMS oI �he bar. This load CMl be. replaced with sufficient aeeinacy by a trnasvere load folkiwing the law .... [231 [ntrodueing the load q in Equation [l ], we ea.o. reduce our r•rnbtem to the evaluation of AJl elliptic integral of the Mat kind ! m [ :;

  The usual theory of vibration ol the btl.t'fl is balle d on the aaaump tion that one end of the lw-, being free to mow in an axial direetio n, an extensio.nle&l deflection of the bar ill obtained . ln t.eelmieal practice we often have to deal with Immovable end binpi, or with hinges � with support.sin wch a manner that, as the end!; approach: each <>ther, e. tensile. foree bf produced in the bar--which. is proportiowi,l t.o the amount of that motion. fn these eases the efrect of the a:xfal force on the prooesB Qf vifm:L..

	tion must. be investigated. Further, we 8llSl ll ll l'l an initial tensile force and an exl:enfii bility of the bar . The deflection of the bt.r

z, of bar E "" Young's modulus of mat.eria.l A. ,.. cr�onal area. of bar d ... El "" flexural rigidity of bar r "" v' I/ A ... radius of gyration fJ ""' spring oonst&nt of suppor\3 of bar relative to &Jdal dieplace-.,.nt s� ... initial it.rial «insile force of bar S1 = axial tenaile fofl:le due to defumtion q = traOS'Verse load pei:-unit length t = l.ime iJ; "" a function oft alone ,.. ... vil:iration mass pet mrit length of bM a = half amplitude of vibration l:l! = a/r "' ... frequency in radians Pfil' ooo n .. pasitive integers P" "" � = Euler's load Qf bar f,,r buckling fonn with n half .. v,, Po ..,. -Soinitial oompr eoo ive load of bar Vnnu,Ttoo OF BiBs dOM not need t.o be om.all in comparison with its transverse 1 :Frankfort-on.•Main, Ge:tmADy.

  F-0, we should.only rephloo the actual area A in our final rewlta by the reduced value

	A '.,.	48A . 1-� "	.	. (41
	Equating now the ExpMlflions (2J and (3}, we get S , ... lt'A �

. . .. . . .... . . . ... . = :t;.;:1 (�) t d.1:

  �tisfied. Using Eq.JMl.tion [6), we get the following egua.tion . [81 �fultiplying this by (d,f,)/(dt) &nd integrating the result 00.tWt1en the l'.WL\imun � deflection (f "" l, [d+J/ldt] .., 0) and a.ny

	tor 1f ;;i. +7"'"'0, . defleeti()n 1/1, we obtain d� n'1t4 a' B (n _ �'lf"•B n't'2Sn) di' = -��,,_ , _
	U\1ing the t1.hbrevia.tions
	•nd Bquation' (9] ...... [101 . . . I 1 ii
	. l 12]
	.... [13\

. beoomes [i±.. j . . 1,.,. tl -'/i �)(k"f,' + l -kl)

d(pt)

The solution of Lb.is eqJMl.tlon is

>/I = cn[p(t + M, kJ .

where k i$ the modulus of the elliplie function and kt a oon:.tant vf integra.tion, which we tl\Jl make zero, Now, hy Equatj on [7], each e.. "CpreBHion of the form . •= u ... a sm -1 -en (pt, k) .. . . . ,.

[14]

with n ""' 1, 2, 3,. . is a pO&Sible oolutkin of Equation [ lJ. But this epti.on iB nonlines.r in y, and for this formal reason a super position of any solut.ioll.8 o{ t.he form, Equation (14), W not p.rae tieable. The obvious meehani ca.1 reeo on for difficul ty in obtain• ing a general solution of the problem ilJ the coupling eff eo'.!t of a.rial forC!ffl resulting from eaeh particular aolution of the form of EqWl.tion

[14]

.

The period of the :function cn(:pt, k) is "" � • J."' 12 a,, . •-.... v'1 -k1 sint,, and the eorresp0 nding frequency

  We have now to eoll.8ider the effect of the initial a.xi.al force s�. At firgt let Su be positiVtl, by Equations [10] and [16] the fre quency w then increases if the value 80 is inerea.sing. Now• Wt Su beeome negative. \Vhile :sol = P� ineress ee , the frequenc:• "' deen"a.Se5, and it vanWbes if Po = P. (1 + 1).

	TABLE I EFFECT OF A\\tPtlTUDl!fON INCREASE OF FREQUENCY 0 0.1 0.2 , .. 0.6 0.8 .. 1 ... 1 1.0008 1.01)38 1.015 t .038 1.0118 • 1U � . 5 2 • ' ..1 .. � 1.080 �:�� t.!l�fl t.976 2.3!i
	HenC\l tile frequency of this forot! i� equM to 2w !l.nd its IlllUi mum value i�
	.... [20[

P � being that Eul11r load which corresponds to the orthogtJUal function y ""' sin (nn)/l .


Jaskirat Singh
email: jaskirat@ualberta.ca

Sandeep Kumar Sahoo

Literature Review on Background Subtraction Model for Object Detection

Keywords: Image Processing, Video Processing, Computer Vision, Pose detection, Machine Learning, Action Recognition, CNN, Fish-eye lenses, Spatially-varying sensing, virtual reality, adaptive training, rehabilitation, virtual games I. I

Background subtraction is widely used technique in computer vision application for object detection and segmentation. Most of the work done on background subtraction considers static camera that identify moving objects by detecting areas in a video that change over time, which is not applicable in real world scenarios like moving camera mounted on the autonomous vehicle.it's a difficult problem due to the motion of both camera and the object.In this paper, we extend the concept of subtracting areas at rest to apply to video captured from a freely moving camera. we approach this problem by exploiting optical flow based background subtraction method.

In this paper [START_REF] Basu Ashraf Elnagarf | Motion detection using background constraints[END_REF], Elnagar and Anup Basu introduced a technique for detecting moving object from a moving camera using background constraints. They considered both an object and camera are moving rigidly with respect to each other as well as the background. The author considered a camera system that mounted on a tilt device that allows rotation and translation. The displacement field at each point in the image is computed. From the above figure, the displacement vector is represented as the sum of translational component and a rotational component. For an active camera, a mapping function is defined to map pixels which corresponds to the same 3D point to the corresponding image plane positions. For each pair of images processed in the image sequence, the image[I(t)] at time t is mapped so as to correspond pixel by pixel with the image[I(t+1)] at time t+1.Let P be a 3D point that is projected on both the images: p(t+1) = [x(t+1),y(t+1)] = [x(t)+ut, y(t)+vt] = p(t) + V Any point lies in the image plane should satisfy the equation. Whereas any point that lies on an independently moving object is unlikely to satisfy the equation. Therefore, detecting moving objects becomes easy depending on this constraint, which can be used for both translation and rotation.

B. Pose recognition using randon transform

In this paper [START_REF] Mandal | Pose recognition using randon transform[END_REF], Meghna Singh, Mrinal Mandal and Anup basu proposed a novel method for the recognition of pose estimation using randon transform. The proposed technique first involves background separation step to distinguish foreground object from background model. Then medial skeleton model is obtained from the resultant binary image by thinning the image. Finally the randon transform is applied to detect the orientation of the skeleton line which are not very localized and are rarely straight lines. So the proposed method threshold the Radon transform coefficient to extract the local maxima. Furthermore, the authors designed an efficient matching algo-Figure 2. Randon Transform rithm, Spatial maxima matching, to recognize the unknown pose by matching it to the known pose. Finally the score is computed between the total no of corresponding match and the no corresponding match. The higher the score, the closer the unknown pose is to the known pose. It is observed that an overall recognition rate of over 87 percent is achieved with leg and arm poses.The promising recognition rates achieved with the parameterized Radon transform emphasize the feasibility of the proposed method.

C. Representation, Analysis, and Recognition of 3D Humans:A Survey

In this paper [START_REF] Turaga | Representation, analysis, and recognition of 3d humans: A survey[END_REF], Stefano,Mohamed, Pavan and Anup basu provided an analysis of recent development of representation of human body in 3D data and organised in a taxonomy the representation methods based on the main characteristics of the 3D shape. They classified the methods based on the main characteristics of the 3D shape they capture. They divided the 3D representation into two sub categories: i) spatial, and ii) Temporal. the spatial categories captures properties of the surface and the volume of the face and body. The spatial modeling extracts information from the model's surface. The solutions for surface representation is that of separating them depending on whether they use extrinsic or intrinsic measurements. Methods used in topological representations extract topological information of the human body and face. Skeleton model is widely used to represent human topology. Various techniques like thinning algorithm are used to extract skeleton from the human body. skeletons were used for computing the 3D transformation between two poses of a shape and aligning objects. Methods used in representation based on keypoints by Zaharescu et al. is meshDOG a 3D feature detector that seeks the extrema of the Laplacian of a scale-space representation of any scalar function. The descriptor is able to capture the local geometric and/or photometric properties.

Human representation requires temporal modeling in order to model the temporal dynamics of the event. Trajectory based representation is used to extract features from each 3D frame and then comparing those sequences of those features. The matrix representation is extracted from the overall sequence or its parts, like covariance or Hankel matrices.Markov models are cases where the dynamics can be modeled as a Markovian process and transitions between states can be used to model complex temporal events.Recurrent neural netowrk(RNN) based model learns dynamic patterns from the temporal data.

D. A Multisensor Technique for Gesture Recognition Through Intelligent Skeletal Pose Analysis

In this paper [START_REF] Rossol | A multisensor technique for gesture recognition through intelligent skeletal pose analysis[END_REF], Nathaniel Rossol, Dr. Irene Cheng, and Dr. Anup Basu proposed a novel technique to improve hand pose estimation accuracy using smart-depth sensor technology for tracking hand gestures. Their technique addresses the occlusion issue using pose estimations from multiple leap motion sensors placed at different viewing angles. A classifier is built and trained in offline mode using a premeasured artificial hand. The model then can be used in real-time to intelligently select pose estimations while still running at a high sampling speed of over 120 Hz. Leap motion sensors provide very high 3-D positional accuracy for hands and fingertips at a very high sampling rate. Irrespective of the underlying hand pose recognition technique or sensor used, occlusion is the main problem when using a single visionbased sensor. Hence they proposed fusing data from multiple sensors placed at different viewing angles and analyzing the skeletal poses directly instead of examining the depth maps. Their multisensor skeletal pose estimation approach is composed of the following steps:

1) They used a trained support vector machine (SVM) model to intelligently determine the optimal pose estimation from an array of sensors. They built this model offline with a training dataset, which uses a feature vector composed of a subset of each sensor's output. 2) Then they converted each sensor's pose output into a global coordinate system so that the poses of all of The experimental results indicated that their multiple sensor approach reduced the total pose estimation error by 31.5% compared to a single sensor. The average pose estimation error (in mm) is calculated from the sum of the Euclidean distances of each fingertip from its ground truth position divided by the number of fingers. Using this metric, the worst-case and bestcase performances were found to be 21.45 and 9.5 mm, their approach has an optimal accuracy of 90.8% on average.

E. Variable Resolution Teleconferencing

In this paper [START_REF] Basu | Variable resolution teleconferencing[END_REF], Dr. Anup Basu, Allan Sullivan, and Kevin Wiebe proposed the usage of Variable Resolution(VR) for image compression, and a prototype teleconferencing system based on VR is introduced.

Videoconferencing is a prime application for VR compression for several reasons:

1) Videoconferencing requires fast compression of images.

2) Image quality is not a high priority.

3) The typical 'talking head' scene provides for an ideal fovea location. 4) VR provides constant compression, suitable for transmission of images over channels with fixed bandwidth. The video conferencing system provides transmission of greyscale images from an image server to a display or viewer process. The server process is responsible for capturing, compressing, and transmitting the image. The display process accepts images, uncompresses and displays them on a screen.

In addition to VR, an intraframe difference encoding routine provides additional compression. The difference between pixels in successive frames is calculated and, only changed pixels are transmitted. Frame rates on the order of 1.5 -2 frames per second were achieved between SPARC stations across an Ethernet. With the compression values obtained (up to 98% with interframe encoding), the network can easily handle much higher frame rates. No special boards are required as the system is based on software. The video conferencing system can cheaply and easily be run on an entire network with no additional cost. Work is currently underway to integrate the motion video with audio and drawing programs. The ultimate goal is to produce a true multimedia system that can function in a generic environment, with little or no additional hardware. Such a system will be cheap, easy to upgrade and maintain, and will be portable across many systems.

F. Videoconferencing using Spatially Varying Sensing with Multiple and Moving Foveae

In this paper [START_REF] Basu | Videoconferencing using spatially varying sensing with multiple and moving foveae[END_REF], Dr. Anup Basu and Kevin James Wiebe proposed a new method for videoconferencing using the concept of spatially varying sensing. Various techniques are discussed for combining information obtained from multiple points-of-interest (foveae) in an image. The Variable Resolution(VR) transform has two parameters that affect the resulting image: compression and alpha which controls the distortion at the edges of the image w.r.t. the fovea. A high alpha value gives a sharply defined fovea with a poorly defined periphery; a small alpha value makes the fovea and periphery closer in resolution. The main issue with the basic VR transform is that the images are not rectangular so some areas of the image must be clipped off, or the image will have unused pixels in the corners. The problem is magnified with high alpha values and when the fovea is not located in the center of an image. Two ways are proposed to fix this issue.

1) Modified Variable Resolution(MVR): Use multiple scaling factors, each scaling factor dependent on the angle theta in polar coordinates. This method maintains the isotropic properties of the original formulae but is relatively complex. 2) Cartesian Variable Resolution (CVR): Simplify the formulae by isolating the vertical and horizontal components. In this method, isotropic accuracy is reduced a little but Computational complexity is significantly decreased.

Sometimes there is more than one area of interest to the observer then a decision must be made to either reduce the resolution around each fovea to compensate or retain additional information for each additional fovea, reducing the compression ratio. The quality of an image then depends on the relative position of multiple moving foveae. Two distinct approaches are cooperative and competitive foveae. cooperative foveae calculates the location of a point in the transformed image w.r.t. each fovea separately. The true location is then found by weighting the two estimated points according to the distance of the original point from the fovea. A higher weight is given to the location calculated using the closer fovea. A unique property of cooperative foveae is the existence of "ghost foveae" between them. If only two foveae exist, then the area of the highest quality in the scene will not only be at these foveae but also along the line connecting the foveae.

In competitive foveae, all foveae compete to calculate the position of a point in the transformed image. The fovea which is closest to any point in the original image will be the one that determines its transformed position.

The videophone component provide transmission of grey scale images from an image server to a display or viewer process. The server process is responsible for capturing, compressing and transmitting the image. The display process accepts images, uncompresses and displays them on a screen. Without using any additional hardware, it provided very high frame rates with very high compression upto (98%) using intaframe encoding. The rate at which it can compress images, especially on machines with limited processing speed, and the high quality present in the foveal region, make it ideal for the multimedia market.

G. Enhancing videoconferencing using spatially varying sensing

Human vision can be characterized as a variable resolution system-the region around the fovea (point of attention) is observed with great detail, whereas the periphery is viewed in lesser detail. In this paper, [START_REF] Basu | Enhancing videoconferencing using spatially varying sensing[END_REF], Dr. Anup Basu, and Kevin James Wiebe presented that spatially varying sensing can indeed be useful in videoconferencing. A system that can incorporate multiple and moving foveae. There are various possible ways of implementing multiple foveae and combining information from them. Some of these alternative strategies are discussed and results are compared. They also show the advantage of using spatially varying sensing as a preprocessor to JPEG. The methods described here can be useful in designing teleconferencing systems and image databases. This paper is an extension of the above two papers [START_REF] Basu | Videoconferencing using spatially varying sensing with multiple and moving foveae[END_REF] and [START_REF] Basu | Variable resolution teleconferencing[END_REF]and they have generalized Multiple Foveae and Enhanced JPEG. The concept of cooperative foveae is generalized by creating weights depending on a certain power of distances to foveae. Ghost foveae disappear as the parameter grows, and the quality of the image in the foveae improves at the expense of quality between the foveae. If the power is high, it performs in a similar way as competitive foveae. Spatially varying sensing can be used very effectively to enhance the performance of JPEG at high compression ratios. After compression using JPEG with quality = 1, 98.55% compression with signal to noise ratio (SNR) root mean square (RMS) of 18.25 is achieved. The result of JPEG enhanced by a VR preprocessor achieves 98.56% compression with the SNR (RMS) of 47.23.

An alternate way of combining VR with JPEG would be to change the quality parameter in JPEG compression depending on the distances from the foveae.

H. Interactive Multimedia for Adaptive Online Education

In this paper [START_REF] Cheng | Interactive multimedia for adaptive online education[END_REF], Dr. Irene Cheng, Dr. Anup Basu1, and Randy Goebel presented a broader view of multimedia education, focusing on future applications. The authors envisioned providing publicly accessible education anywhere, at any time, and to anyone. To realize this vision, they proposed the Computer Reinforced Online Multimedia Education (CROME) framework, which integrates the main components of education, including learning, teaching, and testing, as well as adaptive testing and student modeling. Interactive multimedia items such as Drag and Drop, Logical-Mathematical, Language, and Educational items can help improve learning performance by enhancing user satisfaction and engagement. Multimedia content helps to improve concept representation, which is not possible in conventional MCQ and fill-in-theblank formats. They referred to a curriculum-specific question as an "item", following each example item can be used either in learning or testing depending on the log-on status: a practice or testing session respectively. CROME framework is designed for multimedia education and uses a combination of web-development tools in order to optimize the constrained resources and to provide user satisfaction. The development kits include Java 2D/3D applets, Javascript, Flash, J2ME, PHP, and MySQL. The choice aims at platform and browser independence. Similar to the multiple-choice format, a generic template is designed for each category of items, which share certain similarities. Multiple questions can be generated by altering the content inserted into the template. 3D items are used in Computer Adaptive Testing, a student's performance can be evaluated more accurately by considering partial scores. A parameter-based strategy is a more general approach for assigning initial difficulties to items. They used Math questions as examples to illustrate the concept. They incorporate a Process Analyzer in the CROME framework. The objective is two-fold: to assist students to improve their problemsolving skills using step-by-step hints and instructions, and to assist teachers in monitoring student performance so that proper help can be provided in time. Every response given by a student is recorded for performance analysis and student modeling. The Process Analyzer comes with a graphical user interface that provides an engaging and motivating learning environment. They implemented multimedia item authoring templates which are plug-ins integrated into the basic CROME framework. There are two main design challenges: First, the uniqueness of each innovative item type, and second, the need to provide different processing pipelines for online and offline item creation. These challenges are overcome by separating the interface implementation into easy-to-manage logical components. Although there is research in the literature focusing on certain target areas in education, their framework is unique in the way it incorporates automatic difficulty level estimation, item generation, process analysis, and testing beyond subject knowledge.

I. Alternative models for fish-eye lenses

The human visual system can be characterized as a variableresolution system. Foveal information is processed at very high spatial resolution whereas peripheral information is processed at low spatial resolution. Various transforms have been proposed to model spatially varying resolution. Unfortunately, special sensors need to be designed to acquire images according to existing transforms and they are computationally expensive. Two models (FET and PFET) of the fish-eye transform are presented in [START_REF] Basu | Alternative models for fish-eye lenses[END_REF] and their validity is demonstrated by fitting them to a real fish-eye lens. The variable resolution (VR) transform proposed in this paper, namely the Fish-Eye Transform (FET) is based on the characteristics of fish-eye lenses, making it as easy to implement using off-the-shelf lenses. Previously log-polar transform was used and its main drawback is that it needs specialized hardware to produce high-resolution images in real-time. They considered an alternative image transform, namely the Fish Eye Transform(FET) which is based on a simplification of the complex logarithmic mapping described by Schwartz. One problem of applying Schwartz's method directly is that image continuity across the vertical meridian is lost, and cannot be recovered easily. They used a simplified VR projection method and computed cortical polar coordinates and cortical Cartesian coordinates. The image continuity is preserved but the disadvantage of the simplified mapping function is that it produces strong anisotropic distortions in peripheral regions for large values of the distortion parameter lambda. Two modeling alternatives are evaluated, one using the FET, the other using a polynomial function (PFET) to approximate the distortion. An average of several estimates of the center of distortion is evaluated in order to obtain a better estimate. Using the polynomial function described by the PFET model and a set of N data pairs (T, p t ), the objective of the least-squares method is to minimize an error function x. The problem now is deciding what degree of a polynomial represents accurately the recorded data. A standard way of solving this problem is to use the variance of the fitted model with the observed data. One increases the degree of the polynomial as long as there is a significant decrease in the variance u2. The PFET appears to have a better performance in compensating distortions than FET. Inverse mapping to restore the original image can be obtained using the inverse function of the simplified FET model. There is no simple way to obtain the inverse transform for the polynomial model. In future work, They wished to consider the problem of designing fish-eye lenses that fit a given parametric model and also intend to study the problem of fast and reliable obstacle avoidance using fish-eye lenses.

J. Nose shape estimation and tracking for model-based coding

In this paper, [START_REF] Yin | Nose shape estimation and tracking for modelbased coding[END_REF], Lijun Yin and Anup Basu proposed an efficient method to detect and track the nostril shape and noseside shape which limits the search region and uses the shapeadaptive model as a remedy for the shrinking effect based on individual deformable templates. Individual templates are designed for the nostril and nose-side. There are 3 main steps in the method.

Firstly, the feature regions are limited to certain areas by using two-stage region-growing methods (i.e. global region growing and local region growing). In global regions growing, a large growing threshold is selected in order to explore more skin areas. In local region growing, the region information is estimated in the individual feature areas. The initial seed pixel of the skin is selected in the local area, and a smaller growing threshold can be selected so that a small skin area surrounding the facial organ is detected, and produces as much feature information as possible.

Secondly, the pre-defined templates are applied to extract the shape of the nostril and nose-side as they are important for composing facial expressions. A geometric template is applied to the nostril region, which is a twisted pair curve with a leaflike shape to detect the nostril shape and the nose side has a shape like a vertical parabola.

After the facial feature regions and shapes on various organs are estimated, a 3D wireframe model can be matched onto the individual face to track the motion of facial expressions.

A camera is mounted on an active platform (pan/tilt) to take an active video sequence, which shows a talking person with an unconstrained background and the camera rotation is less than 5 degrees. Nostril and nose-side are among the key features that are detected. The overall performance gives an indication of the capability of the system to detect most nose features accurately (Only 18 / 270 frames showed position error in the nose area beyond 3 pixels)

Figure 1 .

 1 Figure 1.

Figure 3 .

 3 Figure 3. Two-sensor setup

Figure 4 .

 4 Figure 4. Flowchart of proposed Technique

Figure 5 .

 5 Figure 5. Comparision of SNR Ratio between various Methods

Figure 6 .

 6 Figure 6. Flowchart of Video Conferencing System Prototype

Figure 7 .

 7 Figure 7. Flow Diagram

K. A Framework for Adaptive Training and Games in Virtual Reality Rehabilitation Environments

In this paper [START_REF] Rossol | A framework for adaptive training and games in virtual reality rehabilitation environments[END_REF], Nathaniel Rossol, Irene Cheng, Walter F. Bischof and Anup Basu proposed a design for virtual rehabilitation system that uses virtual reality training and games to engage patients in effective instructions on the use of wheel chair and introduced a novel framework based on Bayesian networks for self-adjusting adaptive training in virtual rehabilitation environments. In order to allow clinicians to easily design and modify the environments for the patient, an XML scene node based framework was designed and implemented as a 3D-modelling plug-in tool. Using this tool, clinicians can change the layout of training rooms, adjust the position, scale, and rotation of objects, and even assign physical properties such as object mass.The second challenge of the implemented framework was to have the system automatically assess the patient skills and then it intelligently adapts the training simulation accordingly. The designed approach uses Bayesian networks to attempt to determine patient skill levels. The authors performed one experiment in order to determine if the training system was effective enough in teaching the participants to navigate around the obstacles in an environment faster than those who did not receive virtual training. The time taken to complete the obstacle course for the trained participants is mush faster, 81.5 seconds compared to 104.5 seconds for the non trained group.

L. Generating Realistic facial expressions with wrinkles for model based coding

In this paper [START_REF] Yin | Generating realistic facial expressions with wrinkles for model-based coding[END_REF], Lijun Yin and Anup basu presented a texture updating method for detecting and tracking facial textures in active wrinkle areas and mouth-eye areas. They used deformable template matching method to find the fiducial points on a face and developed mesh matching algorithm to track facial expression. A wire-frame model is first adapted onto the face images to track the movement of the expression. Then the texture of interest(TOI) is estimated based on fiducial points detected, such as corners of facial organs. Finally, the facial texture are synthesized using these texture of interest. The fiducial point tracking and model adaptation are performed automatically.

The adaptation procedure consists of two stages: 1)facial fiducial point estimation that produces a correct matching of a face model with the face that leads to correct matching of facial expressions and thus result in a correct selection of textures of interest.Using the information extracted from head motion and facial features (i.e., fiducial points, eye contour, mouth contour, and face silhouette), a 3D wireframe model can be fitted to the moving face. They developed a so-called "coarse-to-fine" adaptation algorithm using the extended dynamic mesh to implement the model adaptation procedure.

M. Perceptually Guided Fast Compression of 3D Motion Capture Data

In this paper [START_REF] Firouzmanesh | Perceptually guided fast compression of 3d motion capture data[END_REF], A. Firouzmanesh, I. Cheng, and A. Basu proposed a compression algorithm for motion capture, transmission for the interactive online 3D environment, and real-time synthesis on mobile data. The proposed method for the compression technique used here is wavelet coding as it is one of the efficient approaches for encoding multimedia data. The main idea of the compression technique is to select different numbers of coefficients for different channels of data based on the importance of each channel. One of the factors to evaluate animation quality is the viewer's region of interest. In order to locate the high attention regions, the author used the Interactivity-Stimulus-Attention Model that explains how interactivity stimulates immersion of cognitive resources. the processing steps involves normalizing the bone values.Then calculating the variation of each channel using the below formula. Combine the effects of relative bone length and variation to obtain a weighted quantity. Finally compute

N. Segmentation of Arterial Walls in Intravascular Ultrasound Cross-Sectional Images Using Extremal Region Selection

In this paper [START_REF] Faraji | Segmentation of arterial walls in intravascular ultrasound cross-sectional images using extremal region selection[END_REF], Mehdi Faraji, Irene Cheng, Iris Naudin and Anup Basu introduced region detection strategy for segmenting the acquired IVUS image frames and experimented with the feature extraction method called External Regions of Extremum levels can segment the luminal and mediaadventitia borders in IVUS frames. This proposed strategy involves below steps: i)remove the typical artifacts of IVUS frames, such as ringdown effects and calibration squares ii)ROI are extracted and are filtered based on their types iii)Perform region selection preocedure and segment the two regions labelling them as luminal and media iv)Finally, both regions are traced by using contours. The author applied their proposed technique on the publicly available dataset containing 326 IVUS B-mode images. The distance between the resultant extracted features with the original lumen and media are 0.22mm and 0.45mm respectively. The outcome of the experiment showed that the method was able to segment the region of interest with < 0.3 mm HD to the gold standards. Using this method, the segmentation results are pretty accurate and can be applied to analyse the internal structure of human arteries from Intravascular Ultrasound images taken with a 20 MHz catheter probe.

O. Hough transform for feature detection in panoramic images

Mark Fiala, Anup Basu [START_REF] Fiala | Hough transform for feature detection in panoramic images[END_REF] introduced an approach to detect features in panaromic non-SVP(single viewpoint) images using a modified Hough transform. The author proposed a new hough transform parameter space for detecting lines in images formed from non-SVP catadioptric panoramic image sensors. The proposed method locates horizontal line features. By mapping edge pixels to a new two-dimensional (2D) parameter space for each mirror lobe, the existence and location of horizontal lines can be found. The motivation for recognizing horizontal and vertical line segments is for its use in 3D model creation for man-made environments where the majority of line edge features are either horizontal or vertical. The double-lobed catadioptric optical arrangement presented in this paper allows two viewpoints to be captured by one image capture device (camera). Each point in this new parameter space corresponds to a plane in space instead of line. The family of planes which are viewed by a panoramic catadioptric sensor as a curved line have 2D and thus this Panoramic Hough transform space is 2D. Experiment was carried out which involves the below steps:

-For each point in the camera image convert to polar coordinates relative to Ucenter, Vcenter for pixels in the range Rmin-Rmax.

-Add the edge magnitude of each point in the image to all possible mapped points in the Hough image.

-Locate peaks in this Panoramic Hough image and declare the existence of a plane corresponding to Rfall.

The results demonstrated the robust performance of this transform and indicated one likely application being panoramic stereoimagery. R