Marie-Cécile Caillaud
email: marie-cecile.caillaud@ens-lyon.fr

Methods to Visualize the Actin Cytoskeleton During Plant Cell Division

Keywords: actin, cell biology, plant, Fluorescent-tagged reporters, electron tomography, immunogold labeling, Phallotoxins, super-resolution microscopy

Mitosis in plants consists of separating the mother cell in two daughter cells by the centrifugal growth of a new wall. This process involves the reorganization of the structural elements of the cell, namely the microtubules and actin cytoskeleton which allow the coordination, the orientation and the progression of mitosis. In addition to its implication in those plant-specific structures, the actin cytoskeleton, in close association with the plasma membrane, abbors specific patterning at the cortex of the dividing cells, and might act as a signalling component. This review proposes an overview of the techniques available to visualize the actin cytoskeleton in fixed tissues or living cells during division, including electron, fluorescent and super resolution microscopy techniques.

Introduction

The topology of the tissue in the green lineage relies deeply on a precise spatio-temporal control and coordination of division and growth. As plant cells cannot relocate by migration, the orientation of the cell division is brought about by a complex interplay between the cellcycle machinery and other cellular structures, including the microtubule and actin cytoskeletons as well as the plasma membrane. When the cell cycle is progressing toward mitosis, most cellular microtubules together with the actin filaments (F-actin) relocate at the cell's cortex in close association with the plasma membrane forming the preprophase band at the cortical division zone (CDZ). In acentrosomal plant cells, the mitotic spindle allows the separation of the genetic material during metaphase/anaphase transition while the phragmoplast drives the formation of a new cell wall separating the daughter cells in telophase at the earlier defined cortical division site (CDS) [START_REF] Livanos | Division Plane Establishment and Cytokinesis[END_REF]. Microtubule and F-actin cytoskeletons are therefore closely associated and regulated to form and orient these three distinct structures. During this process, the actin cytoskeleton, in close association with the plasma membrane, adopts specific patterning at the cortex of the dividing cells, leading to the formation of F-actin enriched (actin twin peaks) and depleted zone (ADZ, Figure 1). This review proposes an overview of the techniques available to visualize the actin cytoskeleton in fixed tissues or living cells during division, including electron, fluorescent and super resolution microscopy techniques.

Electron tomography

Actin cytoskeleton and its interaction with microtubule cytoskeleton was visualized using chemical fixed plant cells [START_REF] Seagull | The effects of tannic acid on the in vivo preservation of microfilaments[END_REF][START_REF] Hardham | Reorganization of cortical microtubules and cellulose deposition during leaf formation in Graptopetalum paraguayense[END_REF][START_REF] Heath | Oriented cellulose fibrils and the cytoskeleton: a critical comparison of models[END_REF]. However, chemical fixation and dehydration techniques, were later revealed to be inadequate for the preservation of cytoskeleton [START_REF] Tiwari | Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluorescence, and rhodamine-phalloidin[END_REF][START_REF] Lancelle | Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes of Nicotiana alata[END_REF].

The efficacy of the technique of freeze-substitution for the preservation of cytoskeletal details is now widely recognized [START_REF] Tiwari | Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluorescence, and rhodamine-phalloidin[END_REF][START_REF] Tiwari | Cytoskeleton and integration of cellular function in cells of higher plants[END_REF][START_REF] Lancelle | Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes of Nicotiana alata[END_REF]).

Using such approach, the actin cytoskeleton at the cortex of the cell was describe to be associated together with the plasma membrane, but also with the endoplasmic reticulum beneath the cortical microtubules [START_REF] Tiwari | Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluorescence, and rhodamine-phalloidin[END_REF]. More recently, the ultrastructure of the actin cytoskeleton in the PPB in onion cotyledon epidermal cells was achieved using highpressure freezing/freeze substitution techniques. This approach allowed the collection of quantitative information on the number and length of F-actin inside the PPB. Using this approach, F-actin cables ∼170-nm-long were observed to bind and to cross-link microtubules [START_REF] Takeuchi | Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells[END_REF]. Electron microscopic observations of glycerinated Haemanthus cells indicate the presence of actin-F in the kinetochore fibers (Forer and Jackson, 1979). But these data are still debated because the preparative techniques used might have induced changes in actin-F distribution (Schroeder, 1976).

Immunogold labeling of F-actin

Using actin monoclonal antibodies, which gave the finest resolution with immunogold staining, it was shown that F-actin was often seen in the near vicinity of another bundle or microtubule, in an end to side or Y-shaped connection, closely parallel or juxtaposed to microtubules. Actin-F bundle length often reached 10-20 µm (Schmit and Lambert 1988a). The distribution of F-actin cables was later visualized using an immunogold-silver-enhanc ed method and compared with the arrangement of immunogold-stained microtubules in the same cells [START_REF] Molè-Bajer | Three-dimensional localization and redistribution of Factin in higher plant mitosis and cell plate formation[END_REF]). In the dividing endosperm cells of the monocot Haemanthus, Factin was observed on the spindle surface and those present within the spindle are arranged parallel to the long axis of the spindle. Using this approach, F-actin was found to not reorient outside the spindle, while in the polar region, F-actin appears to be compressed by the elongating spindle [START_REF] Molè-Bajer | Three-dimensional localization and redistribution of Factin in higher plant mitosis and cell plate formation[END_REF]. In anaphase, shorter F-actin orients in various directions accumulate at the equator, where some of them are incorporated into the phragmoplast [START_REF] Molè-Bajer | Three-dimensional localization and redistribution of Factin in higher plant mitosis and cell plate formation[END_REF].

Phallotoxins

The biologically active components of poisonous Amanita mushrooms such as phallotoxins are known to be specific for the F-actin [START_REF] Dancker | Interaction of actin with phalloidin:: Polymerization and stabilization of F-actin[END_REF][START_REF] Wieland | Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushroom[END_REF][START_REF] Wulf | Fluorescent phallotoxin, a tool for the visualization of cellular actin[END_REF]. Phallotoxins are low in molecular weight, and thus can penetrate plant cells without prior cell wall digestion, and can also be used with or without fixation before the staining step. The first attempt to use phallotoxin to stain actin in the phragmoplast and cell cortex was unsuccessful [START_REF] Clayton | Actin organization during the cell cycle in meristematic plant cells. Actin is present in the cytokinetic phragmoplast[END_REF]Lloyd et al.1985;Gunning & Wick, 1985). Later, staining the unfixed cells with rhodaminyl lysine phallotoxin (rhodamine-phalloidin) in detergent extraction buffer in carrot suspension cells set up the protocol to visualize the actin cytoskeleton during cell division [START_REF] Lloyd | The role of F-actin in determining the division plane of carrot suspension cells[END_REF][START_REF] Traas | An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus[END_REF]. Using this approach, the organization of F-actin in the PPB, mitotic spindle and phragmoplast was described in various plant species including maize, Allium, Tradescantia and BY2 cells [START_REF] Panteris | Abundance of actin filaments in the preprophase band and mitotic spindle of brick1 Zea mays mutant[END_REF][START_REF] Yasuda | Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells[END_REF][START_REF] Valster | Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection[END_REF]Clayton and Lloyd 1985a). Using the BY-2 cell culture stably expressing a fluorescent reporter for microtubules, the behavior of the F-actin cytoskeleton was assessed by co-staining with rhodamine-phalloidin [START_REF] Hoshino | Roles of actin-depleted zone and preprophase band in determining the division site of higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin[END_REF]. Using this approach, the actin depleted zone (ADZ) which appear before the prophase was reported to appear first from one side of the dividing cell, possibly as a cause of polar cytokinesis, as is often observed in BY-2 cells [START_REF] Hoshino | Roles of actin-depleted zone and preprophase band in determining the division site of higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin[END_REF] and as was reported in Arabidopsis cells [START_REF] Cutler | Polarized cytokinesis in vacuolate cells of Arabidopsis[END_REF]. The ADZ finally extends to the other side of the dividing cell forming a ring at the division plane deprived of F-actin [START_REF] Hoshino | Roles of actin-depleted zone and preprophase band in determining the division site of higher-plant cells, a tobacco BY-2 cell line expressing GFP-tubulin[END_REF].

Immunolabeling in fixed tissues

Immunofluorescence labeling, using a monoclonal antibody developed against actin is often used to visualize F-actin in fixed tissues. Using this technique, the formation of an actin ring at the cortex of the cell in preprophase, the "actin PPB" was visualized [START_REF] Palevitz | Actin in the preprophase band of Allium cepa[END_REF]. Double labeling of both microtubules and F-actin was therefore possible with this method, permitting to correlate the F-actin organization in regards to the known organization of the microtubule rearrangement that occurs throughout division [START_REF] Mccurdy | Reorganization of cortical actin microfilaments and microtubules at preprophase and mitosis in wheat root-tip cells: a double label immunofluorescence study[END_REF][START_REF] Li | The type II Arabidopsis formin14 interacts with microtubules and microfilaments to regulate cell division[END_REF]. Colocalization of F-actin with rhodamine-phalloidin and microtubules with tubulin immunocytochemistry in Triticum uestivum L. cv. Kite showed that F-actin and microtubules colocalized at the PPB [START_REF] Palevitz | Actin in the preprophase band of Allium cepa[END_REF]. As the PPB narrows, the F-actin is also confined to an increasingly restricted zone usually wider than the PPB in Allium [START_REF] Liu | Organization of cortical microfilaments in dividing root cells[END_REF]. In synchronized tobacco BY2 cells, the triple staining of microtubules, F-actin and DNA was performed and allowed a better resolution in time and space on the relationship between the mitosis progression and formation of the PPB [START_REF] Katsuta | The role of the cytoskeleton in positioning of the nucleus in premitotic tobacco BY-2 cells[END_REF]). In wheat root-tip cells, the immunolocalization of actin revealed the absence of F-actin at the cortical division zone [START_REF] Mccurdy | Immunofluorescent visualization of arrays of transverse cortical actin microfilaments in wheat root-tip cells[END_REF]). However, depending on the antibody, such a zone was more or less wide [START_REF] Mccurdy | Immunofluorescent visualization of arrays of transverse cortical actin microfilaments in wheat root-tip cells[END_REF][START_REF] Panteris | Cortical actin filaments at the division site of mitotic plant cells: a reconsideration of the 'actin-depleted zone[END_REF]. Early work using immunolocalization of actin cytoskeleton in rye leaf tissue revealed the presence of a meshwork of actin that concentrated near the end walls that face the spindle poles [START_REF] Cho | Actin in the developing stomatal complex of winter rye: A comparison of actin antibodies and Rh-phalloidin labeling of control and CB-treated tissues[END_REF]. Consistently, immunofluorescence labeling of F-actin revealed a more intense reactivity to the actin antibody in the cell pole facing the spindle than the longitudinal side walls (Figure 1, [START_REF] Baluska | Rearrangements of F-actin arrays in growing cells of intact maize root apex tissues: a major developmental switch occurs in the postmitotic transition region[END_REF][START_REF] Liu | Organization of cortical microfilaments in dividing root cells[END_REF][START_REF] Li | The type II Arabidopsis formin14 interacts with microtubules and microfilaments to regulate cell division[END_REF]. Furthermore, the ADZ is short-lived from metaphase through the anaphase/telophase transition (Clayton and Lloyd 1985b;Kakimoto and Shibaoka 1987a). The presence of actin filaments before phragmoplast formation in endosperm cells suggest that microfilaments could act as a substitute for cell-plate "memory" (Schmit and Lambert 1988a).

Co-visualization of F-actin and microtubules by dual immunolabelling in BY-2 showed that the length of phragmoplast F-actin is shorter than that of microtubules [START_REF] Li | The type II Arabidopsis formin14 interacts with microtubules and microfilaments to regulate cell division[END_REF][START_REF] Yoneda | Disruption of actin microfilaments causes cortical microtubule disorganization and extra-phragmoplast formation at M/G1 interface in synchronized tobacco cells[END_REF]). During telophase, the two zones of aligned cortical F-actin over the ends of the cell gradually disappear and are replaced by new interphase networks [START_REF] Liu | Organization of cortical microfilaments in dividing root cells[END_REF]. Despite the handiness of using phallotoxins and the option of removing possible damage to F-actin from the fixation procedure, it has been questioned whether unfixed cells stained with phallotoxins could provide a misleading picture of F-actin configurations in vivo (Kakimoto and Shibaoka 1987b;[START_REF] Sonobe | Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl Nhydroxysuccinimide ester[END_REF]. Moreover, labeling procedures without fixation cannot be used for double labeling and therefore reduce the possibility to study the dynamics of the F-actin together with the microtubule cytoskeleton, for instance. F-actin staining and immunolabelling approaches proved useful when the study takes place in an organism that cannot be transformed, or in multiple mutants for which crosses could be difficult. In these cases, the staining techniques described above are still used today.

In parallel, advances in live imaging have led to the use of fluorescent-tagged actin-binding domains (ABD) or proteins interacting with the actin cytoskeleton [START_REF] Li | The type II Arabidopsis formin14 interacts with microtubules and microfilaments to regulate cell division[END_REF]). These methods allow us to visualize the dynamics of the F-actin cytoskeleton during plant mitosis in vivo using for example confocal microscopy, tracking system and super-resolution microscopy (Table 1).

Fluorescent-tagged actin reporters

•

The fluorescent-tagged C-terminal part of the mouse Talin (GFP-mTn 2345-2541 )

Talin is a cytoskeletal associated protein that localizes specifically in adherents-type junctions with the extracellular matrix [START_REF] Burridge | Talin: A cytoskeletal component concentrated in adhesion plaques and other sites of actin-membrane interaction[END_REF] and is absent from the plant genome. The amino acids 2345-2541 in the C-terminus of the mouse Talin (mTalin 2345-2541 ) are responsible for its actin-binding capacity [START_REF] Kost | A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes[END_REF]. Unlike the full-length protein, mTalin 2345-2541 is unable to nucleate actin polymerization and was, therefore, use as a reporter to visualize F-actin in plants [START_REF] Kost | Non-invasive F-actin visualization in living plant cells using a GFP-mouse talin fusion protein[END_REF][START_REF] Kost | A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes[END_REF]. In tobacco BY-2 suspension cells, GFP-mTalin 2345-2541 colocalizes with rhodamine-phalloidin [START_REF] Kost | Non-invasive F-actin visualization in living plant cells using a GFP-mouse talin fusion protein[END_REF]. Stable and ubiquitous expression of GFP-mTalin 2345-2541 in Arabidopsis transgenic lines was used to visualize F-actin in all tissues with no apparent phenotypic effects [START_REF] Kost | Non-invasive F-actin visualization in living plant cells using a GFP-mouse talin fusion protein[END_REF]. While using GFP-mTalin 2345-2541 the F-actin behaviors in different cell types and organisms were described in interphasic cells and in pollen tubes of different species [START_REF] Holweg | Capturing in vivo dynamics of the actin cytoskeleton stimulated by auxin or light[END_REF][START_REF] Hoffmann | Dynamic rearrangements of transvacuolar strands in BY-2 cells imply a role of myosin in remodeling the plant actin cytoskeleton[END_REF][START_REF] Kost | Non-invasive F-actin visualization in living plant cells using a GFP-mouse talin fusion protein[END_REF]. To my knowledge, F-actin dynamics during cell division was not analyzed using this reporter. Later study revealed that while GFP-mTalin 2345-2541 does not affect actin polymerization, it inhibits its depolymerization by affecting the actin-depolymerizing factor [START_REF] Ketelaar | The actin-interacting protein AIP1 is essential for actin organization and plant development[END_REF]). These findings led to the dropping of this reporter to study F-actin in plants.

• The fluorescent-tagged Fimbrin

Fimbrin is a cytoskeletal protein associated with F-actin in various metazoan structures, such as in microvilli, microspikes, membrane ruffles, and cell-substrate attachment [START_REF] Bretscher | Fimbrin is a cytoskeletal protein that crosslinks F-actin in vitro[END_REF]. The first plant fimbrin-like gene, AtFIM1, was identified in Arabidopsis (McCurdy and Kim 1998) and since then has been extensively used as a reporter to visualize F-actin cytoskeleton inside plant cells [START_REF] Kovar | Fluorescently-labeled fimbrin decorates a dynamic actin filament network in live plant cells[END_REF][START_REF] Wang | Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C-and N-termini of the fimbrin actin-binding domain 2[END_REF]Sheahan et al. 2004b;[START_REF] Wang | Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots[END_REF][START_REF] Yoneda | Recent progress in living cell imaging of plant cytoskeleton and vacuole using fluorescent-protein transgenic lines and three-dimensional imaging[END_REF]). The Fimbrin 1 actin-binding domain 2 fused to GFP (GFP-fABD2) corresponds to the N-terminal fusion of GFP to the C-terminal half of AtFim1 (aa 325-687), which includes the second ABD and the C-terminal end of Arabidopsis Fimbrin 1 (McCurdy and Kim 1998; Ketelaar et al. 2004a). Compared with GFP fusions to full-length AtFim1, GFP-FABD2 is more visibly labeling the native F-actin organization in Arabidopsis (Sheahan et al. 2004a) and rapidly became a standard to visualize F-actin in planta (Voigt et al. 2005a).

In the tobacco BY-2 cell line stably transformed with a GFP-fABD2, the appearance of a broad F-actin band in the late G2 phase that later separates to form a structure corresponding to the so-called ADZ in mitosis was confirmed [START_REF] Sano | Appearance of actin microfilament 'twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin[END_REF]. Intriguingly, the GFP-fABD2 labeled structure at the cell cortex in mitosis appeared to form two bands rather than the ADZ.

Measurements of fluorescent intensities indicated a GFP-fABD2 distribution that resembled two peaks, and we therefore named the structure actin 'twin peaks' (Figure 1, [START_REF] Sano | Appearance of actin microfilament 'twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin[END_REF]).

The cell plate was shown to form exactly in the middle of the actin 'twin peaks' suggesting that these structures act as a landmark for cytokinesis orientation [START_REF] Sano | Appearance of actin microfilament 'twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin[END_REF]. Live cell imaging in BY2 of the GFP-fABD2 distribution was next monitored together with the endocytic tracer, FM4-64, that labels the cell plate [START_REF] Higaki | Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis[END_REF]. During cytokinesis, GFP-fABD2 accumulates at the phragmoplast and subsequently approaches the expanding cell plate edges [START_REF] Higaki | Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis[END_REF]. In vivo visualization of F-actin in Arabidopsis plants stably expressing 35Spro:GFP-fABD2 showed that mitotic cells have an F-actin-depleted zone around the equator, whereas F-actin accumulates at the plasma membrane which is facing the spindle poles (Voigt et al. 2005a). During cytokinesis, this overall actin polarity is still maintained (Voigt et al. 2005a). Using this reporter, the phragmoplast F-actin could be observed at different stages of cytokinesis (Voigt et al. 2005a).

In Arabidopsis, the expression of the GFP-fABD2-GFP reporter under the 35S promoter [START_REF] Wang | Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C-and N-termini of the fimbrin actin-binding domain 2[END_REF]) has inhibitory effects on cell and organ growth [START_REF] Dyachok | Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: fluorophore variant, actin binding domain, and promoter considerations[END_REF]). The use of the ubiquitin10 (UBQ10) promoter to drive expression of the GFP-fABD2-GFP reporter minimized some of the artifacts that researchers might encounter when using fABD2-based and other F-actin reporters for studying F-actin dynamics in living plants [START_REF] Dyachok | Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: fluorophore variant, actin binding domain, and promoter considerations[END_REF]).

• Lifeact reporter

An important development is the identification of a 17-amino-acid peptide (Lifeact) derived from the N-terminus of yeast Abp140 protein that binds specifically to F-actin with a low affinity and appears to be a faithful reporter of actin dynamics without disrupting them [START_REF] Riedl | Lifeact: a versatile marker to visualize F-actin[END_REF]. While Lifeact decorates F-actin with minimum perturbation of actin dynamics in plants, its expression needs to be optimized to reach a lower expression level than fADB2 in plants [START_REF] Era | Application of Lifeact reveals F-actin dynamics in Arabidopsis thaliana and the liverwort, Marchantia polymorpha[END_REF]). Indeed, It was reported that Arabidopsis seedlings overexpressing at high level Lifeact-YFPv reporter under the 35S promoter had less dense and more bundled F-actin in some cell types compared with fABD2-based reporter [START_REF] Dyachok | Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: fluorophore variant, actin binding domain, and promoter considerations[END_REF].

To test whether Lifeact-psRFP affects the dynamics of actin-F in BY-2 stable line, the cells were challenged with Latrunculin B, a drug which elimines actin-F depending on their that should be helpful in performing colocalization analysis in Arabidopsis.

To validate the use of this reporter to visualize the F-actin in plant, BY-2 cells stably expressing Lifeact-psRFP were PFA-fixed, and stained using Alexa-Fluor®488 labeled phalloidin. This experiment showed a clear colocalization of Alexa-Fluor®488-phalloidin and Lifeact-psRFP for the perinuclear actin. However, in the cell cortex, only the green fluorescence of Alexa-Fluor®488-phalloidin could be detected, whereas the red Lifeact-psRFP signal was absent, indicating that there is a clear structural difference between these actin sub-populations [START_REF] Durst | Organization of perinuclear actin in live tobacco cells observed by PALM with optical sectioning[END_REF].

While studies relying on fluorescent-based reporters multiplied, it is important to keep in mind that these biosensors might not label the entire population of F-actin filaments in cells due to competition with endogenous actin regulatory proteins. As such, accurate detection of F-actin by immunolocalization might be considered as a complementary approach in certain cell types [START_REF] Sadot | The Actomyosin System in Plant Cell Division: Lessons Learned from Microscopy and Pharmacology[END_REF].

MT-actin interactions and how to image both at the same time

Interaction between microtubules and F-actin has been reported in various plant systems, and proteins connecting these two cytoskeletal networks were identified, even if the molecular mechanism is still elusive [START_REF] Takeuchi | Actin-microtubule interaction in plants[END_REF][START_REF] Sampathkumar | Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis[END_REF]SCHMIT and LAMBERT 1988b). Among them, Kinesins with a calponin homology domain (KCHs) have been identified recently as a plant-specific subgroup of the kinesin-14 family and are suspected to act as microtubule-actin filament cross-linkers. The Cotton kinesin GhKCH2, which also contains an actin-binding calponin homology (CH) domain in the N-terminus, decorates the phragmoplast midzone in dividing root tip cells using immunolocalization assays [START_REF] Xu | A cotton kinesin GhKCH2 interacts with both microtubules and microfilaments[END_REF]). Similarly, NtKCH, a KCH homolog of tobacco BY-2 cells, accumulates in the PPB [START_REF] Klotz | A novel actin-microtubule cross-linking kinesin, NtKCH, functions in cell expansion and division[END_REF]. Using the transient Agrobacterium-mediated transformation of BY-2 cells (TAMBY2), the Arabidopsis kinesin 14-type molecular motor (AtKinG) was found to strongly label the PPB [START_REF] Buschmann | Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells[END_REF]. Fluorescence double-labeling in BY-2 cells showed that AtKinG localizes to both microtubules and F-actin [START_REF] Buschmann | Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells[END_REF]). In BY-2 cells, the fluorescent-tagged kinesin-like AtKCA1 protein from Arabidopsis that interacts with the Cyclin-dependent kinases AtCDKA labels the plasma membrane in a non-homogenous manner [START_REF] Vanstraelen | A plant-specific subclass of C-terminal kinesins contains a conserved a-type cyclin-dependent kinase site implicated in folding and dimerization[END_REF][START_REF] Vanstraelen | Cell cycledependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells[END_REF]. In a stable BY-2 line, GFP-AtKCA1 is excluded from the cortical division zone (CDZ) forming the 'KCA1 depleted zone' (KDZ) [START_REF] Vanstraelen | Cell cycledependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells[END_REF]. This finding contrasts with what was observed for the fluorescenttagged actin nucleator FORMIN 6 (AtFH6-GFP) which labeled the plasma membrane without showing regions of reduced fluorescence [START_REF] Vanstraelen | Cell cycledependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells[END_REF]). To visualize F-actin together with the AtKCA1 fusion protein, fABD2 was fused N-terminally to RFP (fABD2-RFP)

and stably transformed in BY-2 cells expressing GFP-AtKCA1 [START_REF] Vanstraelen | Cell cycledependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells[END_REF]. Dual live-cell imaging showed that the ADZ and the KDZ colocalize [START_REF] Vanstraelen | Cell cycledependent targeting of a kinesin at the plasma membrane demarcates the division site in plant cells[END_REF]).

However, the localization of AtKCA1 in its native system was not yet reported and therefore the existence of the KDZ in a dividing plant tissue still needs to be confirmed.

The Myosins which constitute a large superfamily of proteins that share a common domain of interaction with F-actin, were also reported to colocalize with F-actin and microtubules. The most studied of them, the moss Myo8, was shown to interact with both Factin and microtubules. In Physcomitrella, Myo8 localizes to the division site and the phragmoplast during cell division and may promote phragmoplast guidance via F-actin to the division site [START_REF] Wu | Myosin VIII associates with microtubule ends and together with actin plays a role in guiding plant cell division[END_REF].

While microtubule-associated proteins are rarely reported to also binds to F-actin, a 190 KDa Microtubules Associated Protein (NtMAP-190) isolated from mini-protoplasts of Tobacco cultured cells was shown to bind to both F-actin and microtubules and to localized at the mitotic spindle [START_REF] Igarashi | Isolation of a novel 190 kDa protein from tobacco BY-2 cells: possible involvement in the interaction between actin filaments and microtubules[END_REF].

The Formins are involved in the polymerization of actin and associate with the fastgrowing end of the F-actin. In moss, the fluorescent-tagged class II Formin 2A (For2A) strongly labels the center of the expanding phragmoplast, as it was also reported for the type I Arabidopsis Formins AtFH5 and AtFH8 (Ingouff et al. 2005b;Xue et al. 2011a;[START_REF] Wu | Myosin VIII associates with microtubule ends and together with actin plays a role in guiding plant cell division[END_REF]. These observations suggest that the fast-growing ends of F-actin are anchored at its midzone and the pointed ends are in the cytoplasm [START_REF] Wu | Myosin VIII associates with microtubule ends and together with actin plays a role in guiding plant cell division[END_REF].

By contrast, the type II Arabidopsis Formin AtFH14 that, like For2A, acquires an N-terminal PTEN-like domain (van Gisbergen and Bezanilla 2013), colocalizes with a-tubulin at the entire PPB, spindle and phragmoplast when expressed in BY-2 and Arabidopsis suspension cells [START_REF] Li | The type II Arabidopsis formin14 interacts with microtubules and microfilaments to regulate cell division[END_REF]). These finding highlights specificity among the Formins in their interaction with the cytoskeleton and anionic lipids. Indeed, the type I Arabidopsis Formin AtFH4 is able to bind microtubules in vitro and in vivo (Deeks et al. 2010a), suggesting that specific Formins could have an impact on both the F-actin and microtubule cytoskeleton during cell division.

While these fluorescent-tagged actin-binding proteins are available (Table1), the fact that they often localize to both actin and microtubules can make the interpretation of labeling experiments difficult. However, peculiar staining patterns such as the one observed for Myo8 during cytokinesis will be key to study in detail in time and space the events occurring for example at the cortex of the cell in late telophase.

Name

Visualization of F-actin in vivo by super-resolution microscopy

As electron microscopy did in the past, super-resolution microscopy provides the ability to see details of cellular and even macromolecular structures that were not possible to see before. Photo-Activated Localization Microscopy (PALM) has attracted considerable attention because they allow structures below the Abbe limit to be resolved [START_REF] Hedde | Super-resolution localization microscopy with photoactivatable fluorescent marker proteins[END_REF].

In recent years, a growing interest to use this method in plant research is obvious even though

Conclusion

In the past few years, the increasing interest in the visualization of the actin cytoskeleton is illustrated by the number of different techniques and fluorescent reporter lines generated.

Yet, so far, the behaviors of the actin cytoskeleton during mitosis have been mostly studied in a single cell context such as the BY-2 cell line. The dynamics of the actin cytoskeleton in growing tissues remain partial, even if recent studies started to address it. To date, there are still difficulties to visualize dividing cells in the meristematic part of the plant at super-resolution because of the topology of the tissues or the presence of the root cap. Many other technical challenges are still blocking progress in the field, to address for example whether or not the ADZ is also observed in a dividing cell in its physiological context. In a tissue context, the fluorescence observed in the surrounding interphase cells, might mask potential domains at the cortex of the dividing cell such as the ADZ. The development of an inducible promoter or artificial system to visualize the dividing cell at better resolution might be the next task.

Furthermore, while many zonation in the cortex of the dividing cells (CDZ, ADZ, actin twin peaks, KDZ for example) were described in different systems, the temporal and spatial relationship between them is still cryptic. Integration of the localization of the F-actin cytoskeleton in a referential together with the key player of the cell division seems to be the only way to assemble the pieces of the puzzle during this crucial process. in animals, actin

  innate turnover. The authors assumed that If the expression of Lifeact-psRFP increases the stability of actin-F, the cells expressing Lifeact-psRFP should be less sensitive to Latrunculin B. However, the Lifeact-psRFP expressing cell line was more sensitive, which argues against reduced dynamics of actin-F[START_REF] Durst | Organization of perinuclear actin in live tobacco cells observed by PALM with optical sectioning[END_REF].Using Arabidopsis plants, stably expressing Lifeact as a reporter, we tracked the behaviors of the F-actin cytoskeleton in time with an unpreceded precision(Lebecq et al., in prep for Plants 2021 special issue). To follow in vivo division in the root meristem overtime, manual adjustment is needed, otherwise the area under study will grow out of the field of view (the root tip grows out of the field of view in about 20 min). By developing an unsupervised approach to automatically track cells using spinning disk confocal microscopy[START_REF] Doumane | Automated Tracking of Root for Confocal Time-lapse Imaging of Cellular Processes[END_REF], we are now able to automatically track the region under study in real time during acquisition so that root growth can be compensated. This keeps the region under study within frame for the duration of the experiment. By imaging in vivo, the fluorescent-tagged Lifeact under the control of UB10 and RPS5a promoters-both less strong than 35S promoter-we confirmed and quantified earlier reports describing an apical-basal accumulation of the actin filaments in the late stage of cell division (Figure1,[START_REF] Cho | Actin in the developing stomatal complex of winter rye: A comparison of actin antibodies and Rh-phalloidin labeling of control and CB-treated tissues[END_REF][START_REF] Baluska | Rearrangements of F-actin arrays in growing cells of intact maize root apex tissues: a major developmental switch occurs in the postmitotic transition region[END_REF][START_REF] Liu | Organization of cortical microfilaments in dividing root cells[END_REF][START_REF] Voigt | GFP-FABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings[END_REF][START_REF] Sadot | The Actomyosin System in Plant Cell Division: Lessons Learned from Microscopy and Pharmacology[END_REF][START_REF] Doumane | iDePP: a genetically encoded system for the inducible depletion of PI[END_REF] prep for Plants 2021 special issue). By visualizing fluorescent-tagged Lifeact together with cell division markers in the dividing root tip cells, we observed that the F-actin polarization occurs at the metaphase/anaphase transition and persists in time throughout cytokinesis (Lebecq et al., in prep for Plants 2021 special issue). In this study, we generated a set of fluorescent-tagged Lifeact markers in cyan, yellow, and red (Lebecq et al., in prep for Plants 2021 special issue),

  photoactivatable or photoconvertible by irradiation with visible light. By activating them sparsely and stochastically, each CCD camera frame contains only a few spots of the size of the point-spread function, which allows each molecule to be precisely localized.

  mutants have strong phenotypes. It's more difficult to understand the role of F-actin in plants, simply because the phenotypes are weak. It would be therefore helpful to obtain more quantitative data about the localization of the actin during cell division and to develop systems to modify the actin cytoskeleton in order to build up a model in the light of what was done for the microtubules cytoskeleton in plants.

Table 1 . Examples of Fluorescent marker available to visualize F-actin in vivo

 1

Acknowledgement

We are grateful to the SiCE group (RDP, Lyon, France) and to Oliver Hamant (RDP, Lyon, France) and Jan Traas (RDP, Lyon, France for comments and discussions. This work was supported by ANRJC/JC INTERPLAY (ANR-16-CE13-0021) and ANR PRC PlantScape and a SEED FUND ENS LYON-2020.

Figure Legend


Jean Maillet

Jeremy Rossi

François Hug

Jean-Jacques Proquez

Antoine Nordez
email: antoine.nordez@univ-nantes.fr

Influence of experience on kinematics of upper limbs during sewing gesture

Keywords:

To teach a skilled motor task, it is crucial to understand the characteristics of expertise. The aim of the present study was to compare the kinematics of the hand sewing task between novices (n = 10), intermediates (n = 10) and experts (n = 10).

Compared to novices and intermediates, the proximal joint of expert participants was less involved in the task than their distal joints. The shoulder of experts stayed closer to the trunk, while the ranges of motion of the wrist and fingers were higher. This ability enabled them to avoid lifting the arm, which was resting on the table. We observed a low cycle-to-cycle variability of the movement pattern for experts, while it was more variable in novices. Moreover, experts shared similar joints synergies attesting of an "experts" common gesture. This knowledge gained about the hand sewing kinematics can be used to refine the training process of dressmakers.

Introduction

The hand sewing task requires very high control and regularity of the upper limb. The needle must be inserted up to fifteen times per minutes with a 0.2 mm gap between two threads, up to several hours a day. Even though sewing machines progressively replace manual sewing, hand sewing is still needed in luxury factory for specific designs and clothes.

The seamstress population is declining and is hardly renewed. It raises the issues of task back up and training the new seamstresses, which becomes crucial in several luxury factories. Training is long (typically 3-5 years) and traditionally done on the field by experienced seamstresses. No objective test is available to evaluate the skills, and certification is subjective to an experienced seamstress' judgment. Although biomechanical analysis may provide crucial information about the motor performance during skilled tasks [START_REF] Uemura | Analysis of hand motion differentiates expert and novice surgeons[END_REF], it has not yet been applied to hand sewing.

Hand gestures have been compared between novices and experts in various working fields such as surgery (McNatt et Smith 2001;[START_REF] Lin | Objective skill evaluation for laparoscopic training based on motion analysis[END_REF][START_REF] Megali | Understanding expertise in surgical gesture by means of Hidden Markov Models[END_REF] manutention jobs [START_REF] Plamondon | Lifting strategies of expert and novice workers during a repetitive palletizing task[END_REF][START_REF] Min | The effects of safety handrails and the heights of scaffolds on the subjective and objective evaluation of postural stability and cardiovascular stress in novice and expert construction workers[END_REF] and music playing [START_REF] Fernandes | Grip pattern and finger coordination differences between pianists and non-pianists[END_REF][START_REF] Parlitz | Assessment of dynamic finger forces in pianists: effects of training and expertise[END_REF][START_REF] Wiley | Cortical activations in primary and secondary motor areas for complex bimanual movements in professional pianists[END_REF]. These studies showed differences in kinematics with experts displaying more stable and reproducible movement [START_REF] Uemura | Analysis of hand motion differentiates expert and novice surgeons[END_REF][START_REF] Fernandes | Grip pattern and finger coordination differences between pianists and non-pianists[END_REF][START_REF] Sakakura | Biomechanical profiles of tracheal intubation: a mannequin-based study to make an objective assessment of clinical skills by expert anesthesiologists and novice residents[END_REF]. In addition, experts have a specific coordination (Fernandes et Barros 2012) that allow them to avoid unnecessary movement [START_REF] Zhou | Qualitative and quantitative assessment of technical skills in percutaneous coronary intervention: in vivo porcine studies[END_REF][START_REF] Hofstad | A study of psychomotor skills in minimally invasive surgery: what differentiates expert and nonexpert performance[END_REF][START_REF] Azari | Quantifying surgeon maneuevers across experience levels through marker-less hand motion kinematics of simulated surgical tasks[END_REF] and to minimize muscle force [START_REF] Parlitz | Assessment of dynamic finger forces in pianists: effects of training and expertise[END_REF]. In general, experts display a better efficiency owing to an optimization of the energy expenditure and time [START_REF] Lin | Objective skill evaluation for laparoscopic training based on motion analysis[END_REF][START_REF] Plamondon | Lifting strategies of expert and novice workers during a repetitive palletizing task[END_REF][START_REF] Azari | Quantifying surgeon maneuevers across experience levels through marker-less hand motion kinematics of simulated surgical tasks[END_REF]. For [START_REF] Lin | Objective skill evaluation for laparoscopic training based on motion analysis[END_REF], a more efficient movement during laparoscopic surgery is linked to a relative use of joints, specific to expertise: larger range of motion for distal joints compared to proximal joints. Therefore, a kinematic analysis seems relevant to better understand the performance of experts in an upper limb task that requires a high level of accuracy like hand sewing. However, kinematic analysis on the upper limb could be quite complicated considering the numerous degrees of freedom. To simplify this analysis and to better understand the time changes in joint angles, a principal component analysis (PCA) could be relevant [START_REF] Santello | Patterns of hand motion during grasping and the influence of sensory guidance[END_REF]. It provides synergies at the joint level that may be specific to expertise.

The aim of the present study was to compare the kinematics of hand sewing between novices, intermediates, and experts. Our goal is to better understand the characteristics of expertise for this task and to contribute to the task back-up and training. In relation to the state of the art on other upper limb tasks, we hypothesized that experts will produce specific movement patterns with lower proximal joint ranges of motion and larger distal joint ranges than intermediates and novices.

Methods

Participants

Three groups of participants were studied (total: 30 participants). The first group was composed by ten experts (age: 42.5 ± 14.2 years, height: 1.64 ± 0.08 m, weight: 68.6 ± 13.4 kg, 10 females) with a strong experience in hand sewing (14 ± 10 years, range 3-35 years). They were recruited from two French luxury companies. The amount of weekly practice in hand sewing was about 29.5 ± 5.5 h. The second group was composed by ten intermediate -dressmakers' students (age: 18 ± 2 years, height: 1.62 ± 0.06 m, weight: 54.8 ± 7.5 kg, 10 females) with a small experience (3-6 months). The control group was composed of ten novices with no experience (age: 24.8 ± 3.3 years, height: 1.73 ± 0.08 m, weight: 64.1 ± 10.6 kg, 5 females and 5 males). Participants' skill level was determined by their experience with sewing, we can note that expert group was compose of professional seamstress. Twenty-seven participants were right-handed and three were left-handed (one in each group). None of the participants had an upper-arm injury in the last 6 months. The experiment was approved by local ethics committee of Nantes University (n • 20032020-1) and each participant signed an informed consent.

Experimental task

The experimental session was designed as three set. The sewing cycle was repeated ten times for each set (48 ± 15.8s by set), separated by 1 min of rest. The experimental session, including the anatomical markers disposition lasted about 45 min. The type of stitch sewing used for the experimental task was the "Whip stitch" (Appendix 1). The non-dominant hand presented the tissue (100% cotton tissue) by the indexthumb pinch, the dominant hand holds the needle (no.10) by the index-thumb pinch. The task consisted in a sewing cycle composed by three phases (Fig. 1). First, participants placed the needle against the back of the tissue in the right place to introduce it precisely (Approach phase). Second, participants introduced and pushed the needle through the tissue and grip again the needle (needle-tissue contact phase). The needle-tissue contact phase was determined like the cycle moment when the distance is minimal between markers (LM2 and RM2: placed on the distal head of the 2nd metacarpal) of the left and the right hand, and until the distance between them increase (+3 mm). Third, participants pulled it to stretch the sewing thread (polyester thread no.120) (Pull thread phase). Participants were seated to an adjusted table, in such way that forearms lied on the table, the back was straight, arms were close to the body and elbows were flexed at 90 • . Participants were required to sew following a line as close as possible with a gap between each stitch to 2 mm-4 mm. In addition, they were required to sew with fluidity without time constraint. Before each session the thread was cut at 40 cm. Sewing cycle started and finished when the dominant hand was at the highest position (the end of the pull thread phase).

Kinematics

During each of the three sets, kinematic data were recorded at 100 Hz with a system composed of twelves motion capture cameras (Flex 13, Optitrack, Corvallis, Oregon, USA) placed around the participant, six were focus on fingers area (40 × 40 × 40 cm), others recorded body movement in general. Fifty-two spherical reflecting markers (4 mm in diameter) were placed on anatomical landmarks of upper-limb (Fig. 2) [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion-Part II: shoulder, elbow, wrist and hand[END_REF][START_REF] Goislard De Monsabert | Quantification of hand and forearm muscle forces during a maximal power grip task[END_REF]. Marker locations are described in supplemental data (Appendix 2).

We recorded kinematic data for 21 joints with a total of 36 degrees of freedom (DoF). Three-dimensional position of the chest, arms, forearms, hands and the first, second and third fingers for dominant hand and only the first and second fingers for non-dominant hand were considered during the movement using the kinematic model [START_REF] Wu | ISB recommendation on definitions of joint coordinate systems of various joints for the reporting of human joint motion-Part II: shoulder, elbow, wrist and hand[END_REF][START_REF] Goislard De Monsabert | Quantification of hand and forearm muscle forces during a maximal power grip task[END_REF][START_REF] Fohanno | Improvement of upper extremity kinematics estimation using a subject-specific forearm model implemented in a kinematic chain[END_REF]. Each bony segment was located by three markers and a local coordinate system was created (Fig. 3). The longitudinal axis linked two joint centers oriented proximally (Y for upper-limb, X for the hand). A plan was created with the third marker of the segment, placed on the flexion/extension (Flex/Ext) axis of joint. The Frontal axis (Adduction/Abduction; Add/-Abd) was created perpendicular to the plan oriented frontally (X for upper-limb, Y for the hand). Finally, the transversal axis (Flex/Ext, Z) was orthogonal to both X and Y axes, pointing right (right/radial or ulnar/left hands). Since only Flex/Ext around transversal axis was evaluated for fingers, only two markers were placed on finger phalanges. Two additional markers were placed on the thumb to compute internal and external rotation (X axis) [START_REF] Cooney | The kinesiology of the thumb trapeziometacarpal joint[END_REF]. Joint angles were calculated as the product from orientation matrix of proximal segment regarding the distal segment [START_REF] Cappozzo | Human movement analysis using stereophotogrammetry: Part 1: theoretical background[END_REF], and owing to Euler's angles according to rotation sequence: Z (Flex/Ext), X (Add/-Abd), Y (Pronation/Supination; Pron/Sup) around fixed axis. Joint angles obtained during static capture in reference anatomical position, considered like zero angle, were subtracted from angles computed during movement. All calculations were performed using custom-made Matlab scripts (2019b, Mathworks Inc, USA).

The limb that holds the tissue was almost static. We focused on the dominant hand and arm of the participants to analyze 20 DoF on 12 joints. Only the best set of ten cycles, the more regular without parasite movement, were kept then averaged to provide a representative cycle. The three phases were interpolated on 100 points for each participant, then the entire cycle was time normalized from 0 to 100% of the cycle.

Data reduction

The principal component analysis (PCA) method was used to reduce the DoFs redundancy and identify motor synergies, which describes the covariation of joints' motion during the task (Lambert-Shirzad et Van der Loos 2017). PCA consists of an orthogonal transformation that converts the variables into new uncorrelated principal components, which explain the majority of variation in the data (Deluzio et Astephen 2007). The principal components are arranged in order of their decreasing sample variance. For each principal component, the PCA results provide a coefficient and associated load vectors: the coefficient represents the temporal pattern of the principal component, whereas the load vectors represent the weights of each variable within this component.

For each participant, we mean-centered the kinematic data of the ten sewing cycles for each of the 20 DoF. Last, we applied eigenvalue decomposition of the covariance matrix. We considered the principal components needed to explain 90% of the variance of the original dataset [START_REF] Jackson | A User's Guide to Principal Components[END_REF].

Statistical analysis

To analyze differences in sewing cycle duration (s) and relative needle-tissue contact phase duration (s) between groups, two two-way ANOVAs with post-hoc (Bonferroni) were performed. (within subject factor = start and end of the needle-tissue contact phase; betweensubject factor = Group).

The method of statistical parametric mapping (SPM, [START_REF] Pataky | Zero-vs. one-dimensional, parametric vs. non-parametric, and confidence interval vs. hypothesis testing procedures in one-dimensional biomechanical trajectory analysis[END_REF] was used to assess differences in joint angles between the groups through the movement cycle. Specifically, to locate the differences of joint angles across the three groups, we used a one-dimension SPM ANOVA with post-hoc (Bonferroni) for each DoF.

To test the between-group difference in range of motion, a separate one-way ANOVA with post-hoc (Bonferroni) was performed for each DoF.

Coefficient of multiple correlation (CMC, [START_REF] Ferrari | A new formulation of the coefficient of multiple correlation to assess the similarity of waveforms measured synchronously by different motion analysis protocols[END_REF] was calculated to assess the variability of the time-varying joint angle profiles across participants (between-subject variability) and across cycles (within-subject variability) for each DoF. Then, to compare CMC between groups, a one-way ANOVA with post-hoc (Bonferroni) was performed for each DoF.

Finally, to compare the number of principal components that accounted for 90% of variance between groups, a one-way ANOVA with post-hoc (Bonferroni) was performed. To compare the load vectors of each principal components between the three groups, one two-way ANOVA with post-hoc (Bonferroni) was carried out (within subject factor = twenty principal components; between-subject factor = Group). To evaluate the similarity of the component's coefficient between participants of a group, a CMC was calculated for the three principal components. To evaluate the correlation of vector load between participants of a group, a Pearson correlation was performed for the first principal component. Then, to compare principal component's vectors loads correlation between groups, a one-way ANOVA with post-hoc (Bonferroni) was carried out.

Statistical analysis was carried out with Matlab (2019b) and Statistica (v10, StatSoft, France). The significance threshold was set at p = 0.05.

Results

There was a main effect of group (p = 0.001) on the duration of the sewing cycle, with the cycle being shorter for experts (3.4 ± 0.8s) than for both intermediates (5.6 ± 1.6s; p < 0.003) and novices (5.5 ± 2.2s; p < 0.005). There was no difference between intermediates and novices (p = 1). The needle-tissue contact phase (second phase) lasted to 0.83 ± 0.08s (25.5 ± 4.5%) to 2.40 ± 0.62s (71.2 ± 4.4%) for experts, to 1.23 ± 0.44s (21.6 ± 2.7%) to 3.97 ± 1.25s (70.9 ± 5.8%) for intermediates, to 1.03 ± 0.16s (20.0 ± 5.8%) to 3.77 ± 0.86s (68.3 ± 3.6%) for novices, of the total cycle. There was no difference between groups.

The SPM analysis revealed significant differences between groups for five DoF (Fig. 4). Specifically, the mean shoulder abduction joint angle was smaller for experts (16.0 ± 0.4 • ) than novices (36.4 ± 2.1 • ; p < 0.006) over the entire cycle (1-100%), but no differences with intermediates (22.2 ± 1.5 • ) was observed. Middle-MCP extension was higher in experts than both novices between 11% and 83% of the cycle (p < 0.001) and intermediates between 22% and 45% (p < 0.001). First-MCP extension was higher for experts than novices between 1% and 16% and between 89% and 100% of the cycle (p < 0.03). No differences between intermediates and either experts or novices were observed. Fig. 3. Position and orientation of the segment coordinate system of upper limbs and trunk. Blue axis is for flexion (Z), green is the rotation axis for upperlimb and adduction axis for hand (X), and red is the adduction axis for upperlimb and the axis rotation for hand (Y). In grey the frontal plan from landmarks. Middle-PIP flexion was higher for experts than novices from 44% to 60% of the cycle (p < 0.03), no differences between intermediates and either experts or novices were observed. Middle-DIP flexion was higher for experts than novices from 16% to 58% of the cycle (p < 0.001), no differences between intermediates and either experts or novices were observed.

The range of motion of the 20 DoF are depicted for each group in Table 1. For the sake of clarity, only the main effect of group is depicted in the table, while the results of the post hoc tests are depicted in the main text. The range of motion of Shoulder-Flex/Ext was approximately 49% smaller for experts than novices (p = 0.048). The range of motion was larger in experts than novices for three others DoF: Middle-MCP-Flex/Ext (+84%; p = 0.006), Wirst-Add/Abd (+97%; p = 0.002) and Wrist-Pron/Sup (+49%; p = 0.034). The range of motion was larger in intermediates than novices for two DoF: Wirst-Add/Abd (+88%; p = 0.003) and First-MCP-Flex/Ext (+47%; p = 0.045) (Table 1).

The CMC analysis revealed that some experts' DoFs motion were similar between individuals. For 6 DoF we found CMC values > 0.4, revealing a low between-subject variability: Wrist-Pron/Sup (0.41), Wirst-Add/Abd (0.60) (Fig. 5), First-MCP-Add/Abd (0.44), First-MCP-Flex/Ext (0.43), First-PIP-Flex/Ext (0.50) and First-DIP-Flex/Ext (0.43). For intermediates we found 3 DoF with a CMC over 0.4: First-PIP-Flex/Ext (0.56), First-MCP-Flex/Ext (0.45) and Thumb-TMC-Add/ Abd (0.43). None of the CMC values were above 0.4 for novices, revealing a large between-subject variability for all the DoF.

The between-subject variability of the time-varying joint angle profiles was different between groups (main effect p = 0.02). Specifically, a lower variability was found in the expert group (CMC 0.65 ± 0.14) compared to the novice group (CMC 0.49 ± 0.13; p < 0.02). No significant difference was observed between intermediates (CMC 0.52 ± 0.10) For principal components analysis, there was a main effect of group on the number of components needed to reach 90% of the variance accounted force (p = 0.007). Specifically, less components were needed for experts (4.3 ± 1.15) than intermediates 5.8 ± 1.03 (p = 0.013) and novices 5.7 ± 1.06 (p = 0.022) (Fig. 6). No differences were found when we compared the vectors loads between groups for each component (p = 0.449).

Regardless of group, the first synergy explained a large part of the accounted variance: 58.9 ± 11.2% for experts, 51.9 ± 13.9% for intermediates, and 45.4 ± 8.3% for novices. We calculated the CMC value for the coefficient of the three first synergies to assess their variability between participants. CMC values reveal a low between-subject variability for the first synergy: experts (0.93), intermediates (0.88), and novices (0.70). CMC values for experts' coefficients of the three first principal components were high, while the CMC values for intermediates and novices was just high only for the two first principal components and lower than experts score (Table 2

.).

Pearson's correlation for vector load of the first synergy was higher between experts 0.76 ± 0.06, than intermediates 0.55 ± 0.15 (p < 0.05) and novices 0.18 ± 0.27 (p < 0.001). The intermediates score was also higher than novices (p < 0.001) (Fig. 7).

Discussion

Using a kinematic analysis of 21 upper-limbs joints, we observed significant differences in task kinematics between experts, intermediates and novices during a hand sewing task. In accordance with our hypothesis, expert seamstresses exhibited lower proximal joint range of motion and larger distal joint range of motion compared to intermediates and novices. Experts used their shoulder "Shoulder-Add/ Abd & Flex/Ext" less and their arm stayed closer to the trunk. Conversely, they used more of their wrist "Wirst-Add/Abd & Pron/Sup" and fingers "Middle-MCP-Flex/Ext". In addition, expert seamstresses took less time to complete the task and exhibited a lower cycle-to-cycle variability than novices.

The reduced task duration found in the present study clearly demonstrates the better performance of experts. It is in accordance with the literature which focused on skilled motor tasks such as surgery [START_REF] Hofstad | A study of psychomotor skills in minimally invasive surgery: what differentiates expert and nonexpert performance[END_REF][START_REF] De Laveaga | Ergonomics of experts and experts during simulated endotracheal intubation[END_REF][START_REF] Azari | Quantifying surgeon maneuevers across experience levels through marker-less hand motion kinematics of simulated surgical tasks[END_REF]. [START_REF] Azari | Quantifying surgeon maneuevers across experience levels through marker-less hand motion kinematics of simulated surgical tasks[END_REF] showed that the reduction of task duration is obtained by "reducing extraneous movements" and is often associated with an economy of energy. [START_REF] Lin | Objective skill evaluation for laparoscopic training based on motion analysis[END_REF], indicated that experts' efficiency is associated with a specific relative use of joints and showed a larger range of motion for distal than proximal joint. Finger dexterity, defined as the ability to accurately and rapidly control and coordinate finger movements [START_REF] Carment | Manual dexterity and aging: a pilot study disentangling sensorimotor from cognitive decline[END_REF], is preponderant for accurate hand tasks. In the present study, we observed that experts used their fingers differently than intermediates and novices. Specifically, during the phase of the needle-tissue contact phase, the middle-finger joints displayed a dramatically higher range of motion in Flex/Ext (+84% for Middle-MCP-Flex/Ext for experts compared to novices). Our results also highlight the important role of the wrist, showing a greater range of motion for the Pron/Sup and Add/Abd for experts. The action of the wrist allows experts to appropriately orient the hand for an optimized fingers action, like place and manipulate the needle. Middle-finger and wrist, with their combined range of motion, are used to push the needle through the tissue, then to pull the thread efficiently during the Pull thread phase, without additional movement of elbow and shoulder. This ability, shared sometimes with intermediates, to favor middle-finger and wrist action rather than shoulder abduction or flexion avoid lifting the arm mass, which stayed close to the body. This could contribute to reduced energy cost and muscle work.

Previous studies showed that experts such as pianists exhibit better regularity and reproducibility during a common precision hand task, as well as greater finger coordination [START_REF] Fernandes | Grip pattern and finger coordination differences between pianists and non-pianists[END_REF]. The better control of movements was associated with a reduced muscle activation [START_REF] Wiley | Cortical activations in primary and secondary motor areas for complex bimanual movements in professional pianists[END_REF]. In our case, a lower cycle-to-cycle variability of the movement pattern was observed for experts compared to novices. In addition, the principal component analysis showed that experts movement required 4.3 ± 1.1 synergies to explain 90% of variance while intermediates and novices required 5.8 ± 1.03 and 5.7 ± 1.06 synergies. This lower number of synergies in experts can be interpreted as a higher dimensionality reduction of the control of the large number of upper limb degrees of freedom (Schieber et Santello 2004). This ability, acquired with long-term practice and sensorimotor feedback (Sobinov et Bensmaia 2021), leads to the initial complex joint coordination to simpler, repeatable, and efficient movement. Joint ranges of motion ( • ) of 20 DoF Labels for groups of experts (n = 10), intermediates (n = 10) and novices (n = 10). Significant difference between experts and novices (*), between intermediates and novices (#), between experts and intermediates ( †). Adduction/Abduction (Add/Abd), Distal interphalangeal (DIP), Flexion/ Extension (Flex/Ext), Metacarpophalangeal (MCP), Pronation/Supination (Pron/Sup), Proximal interphalangeal (PIP), Trapeziometacarpal (TMC).

For many authors (Nelson, 1983;Yao et Billard 2019), a skilled movement satisfies conditions such as "economy of effort" or "efficiency", and these characteristics rely on an optimal joint movement pattern. In our case as we have shown, an expert seamstress uses less of their proximal joints (Shoulder-Add/Abd & Flex/Ext) and more their distal joints (Wirst-Add/Abd & Pron/Sup; Middle-MCP-Flex/Ext) as compared to novices, characteristics probably linked to an "efficiency" of movement. Experts shared some joint movements patterns like Wirst-Add/Abd and Wirst-Pron/Sup, or First-fingers joints DoFs (0,4< CMC <0,6). Moreover, as shown by the principal component analysis, the three first synergies are very similar between experts, attesting to an "efficient" common gesture. Fig. 8 describes these most relevant differences between groups that seem to determine the hand sewing performance and could be a target to the task learning. It probably corresponds to an optimal movement for this given task and could be used as training basis for the novice and intermediate seamstress. As suggested by [START_REF] Azari | Quantifying surgeon maneuevers across experience levels through marker-less hand motion kinematics of simulated surgical tasks[END_REF], we could consider that novices learn to complete the task, intermediates learn to move faster, and experts learn to conserve energy by reducing extraneous movements (shoulder use).

With training, the movement is optimized and results in better motor control and coordination.

Expert participants were able to avoid lifting their arm, which was resting on the table. In addition, the shoulder range of motion was lower for the experts' group. This behavior is probably crucial to limit the fatigue induced by the task. Thus, it is also probably important to perform the task in that way to prevent MSDs. However, experts bend and twist their wrist further and faster than novices. It can lead to high loads repeated several times during the working days and can expose workers to risk of MSDs like carpal tunnel syndrome [START_REF] Palmer | Carpal tunnel syndrome and its relation to occupation: a systematic literature review[END_REF]. In addition to the wrist motion, a precision (pinch) grip is required. According to Finneran et O'Sullivan (2013), forceful gripping, precision tasks and deviated wrist postures are a combination of factors leading to MSDs. Taken all together, these factors may lead to carpal tunnel syndrome [START_REF] Abbas | Worksite and personal factors associated with carpal tunnel syndrome in an Egyptian electronics assembly factory[END_REF][START_REF] Palmer | Carpal tunnel syndrome and its relation to occupation: a systematic literature review[END_REF][START_REF] Dianat | Musculoskeletal symptoms among handicraft workers engaged in hand sewing tasks[END_REF]). However, it seems important to observe that the tension to insert the needle through the tissue was low. It would be different with another materials like leather. In the present study, it was not possible to isolate risk factors among the experts' participants. For that purpose, the protocol and the method used could be used in a future prospective study to investigate links between the kinematic of the task and the occurrence of MSDs.

The type of stitch sewing used for the experimental task was the "Whip stitch" (Appendix 1). Even though it is rarely used in the professional context, it is easier to perform, which was an important aspect to make comparison between groups. Moreover, it is representative of many types of stitch sewing allowing to generalize our results for others type of hand stitch sewing. Nevertheless, the particularity of the luxury hand sewing is the accuracy of the task and the quality of the tissue, which is generally thin. This context induces specific characteristics of expertise that are evaluated in the present study. However, if different materials such as leather are used, tools and applied forces would probably be different. The decisive factors of task skill would change, causing probably different characteristics of expertise and different joint relative use. To respond to higher forces, the weak joints of fingers would be less used, the type of grasp may change to be 'power grasp' involving the third and little fingers [START_REF] Cutkosky | On grasp choice, grasp models, and the design of hands for manufacturing tasks[END_REF]) and the wrist would be stabilized and used like force transducer (Finneran et O'Sullivan 2013). Nevertheless, we think that some characteristics of expertise would still be present for harder materials: fewer variable cycles [START_REF] Sakakura | Biomechanical profiles of tracheal intubation: a mannequin-based study to make an objective assessment of clinical skills by expert anesthesiologists and novice residents[END_REF], faster movement or reducing unnecessary movements [START_REF] Plamondon | Lifting strategies of expert and novice workers during a repetitive palletizing task[END_REF] leading to simpler movement in respect to a PCA analysis. Therefore, while we speculate that our results would be generalizable to the tasks of seamstress, it could be very relevant to duplicate the present study to better understand how they could adapt their gesture to harder materials. This study requires consideration of some limitations. On the one hand regarding our research design. First, the expertise level was evaluated by the level of experience. We use no other tools to subjectively evaluate the level of expertise. Nevertheless, in our case the level of expertise were sufficiently links to the experience to correctly differentiate the participants in three separated groups. Second, the level of precision was not evaluated during the experimental task. Participants had to follow a line as close as possible with a gap between each stitch to 2 mm-4 mm, the instruction was the same for each group. The precision was not straightforward to assess and, to our knowledge, there is no way to measure it. Third, the needle was held only with the dominant hand and only 3 over 30 participants were left-handed. Therefore, the applicability of our results to left-handed could be debated. Even if we know that right-handers showed a significant asymmetry of topological cortical network properties to privilege right hand movement (Andersen et Siebner 2018), their cortical surface area of regions related to manual motor control is similar to left-handers (Andersen et Siebner 2018) and their dominant manual dexterity induced is not necessarily different. Then, our results could be extended for left-handed participants, and we do not think that the participation of left-handed in our experimental design influence our results. However, our findings are not generalizable to both dominant and non-dominant hand due to the dexterity difference between both hand (Walker et Henneberg 2007).

On the other hand, we must consider some limitations regarding the measuring device. First, despite twelves cameras placed around the participant (among 6 focused on fingers), markers placed on fingers were sometimes hidden by the tissue or the hands. When this occurred, we rebuilt the makers trajectory with a "rigid body rebuilding". While this event occurred during almost all acquisitions, it was on very short time (<10 frames). Therefore, considering the good accuracy of the rigid body rebuilding method [START_REF] Biryukova | Kinematics of human arm reconstructed from spatial tracking system recordings[END_REF], we are confident that this issue did not influence our results. Second, markers were placed on an anatomical landmark. Thus, skin movement can lead to a displacement of markers during movement (radio-ulnar movement) and sometimes do not exactly represent the reality, and this is a common limit for kinematic analysis [START_REF] Ryu | Analysis of skin movement with respect to flexional bone motion using MR images of a hand[END_REF][START_REF] Laitenberger | Refinement of the upper limb joint kinematics and dynamics using a subject-specific closed-loop forearm model[END_REF]. However, it likely occurred in all the participants making the comparison between population meaningful. We therefore believe that it did not affect the main conclusions of our study. Third, more generally, the kinematic methods for the whole upper limb and hand were developed and validated in previous studies [START_REF] Laitenberger | Refinement of the upper limb joint kinematics and dynamics using a subject-specific closed-loop forearm model[END_REF]; [START_REF] Fohanno | Estimating joint kinematics of a whole body chain model with closed-loop constraints[END_REF][START_REF] Goislard De Monsabert | Quantification of hand and forearm muscle forces during a maximal power grip task[END_REF]. This method has already been used for several other applications like paddling [START_REF] Fohanno | Estimating joint kinematics of a whole body chain model with closed-loop constraints[END_REF]. While we acknowledge the classical limitations of our kinematic model (i.e., estimation of joint center locations) (Begon et Lacouture 2005), we think that the hand sewing task is particularly adapted to the use of this model that enabled us to make appropriate comparisons between groups. Within the model used, the thumb is probably the most complicated segment, there is a lack of consensus in the literature for the model of the thumb to be used. We used the method of [START_REF] Cooney | The kinesiology of the thumb trapeziometacarpal joint[END_REF] to analyzed trapezoid metacarpal joint movement. Although it was considered as valid for Flex/Ext and Add/Abd, it may contains inaccuracy for rotation [START_REF] Domalain | Modélisation biomécanique de la main pour l'estimation des contraintes du système musculo-squelettique lors de la préhension pouce-index[END_REF]. It may have influenced our results and this degree of freedom should be interpreted with more caution.

Conclusion

The study was designed to provide better understanding of hand sewing to improve the task back-up and training. Our kinematic analysis showed the importance of posture and relative use of joints. Thus, it seems crucial to keep the arm close to the body and use more wrist and fingers joints rather than the shoulder and elbow. Efficient and fast joint movement shared by expert seamstresses were highlighted (Fig. 8) and can be used as learning targets to improve stitch sewing performance. Future studies would analyze the neural control associated to the kinematic that is used by experts to obtain the specific coordination and finger dexterity highlighted in the present study. In addition, the method developed would be used in a prospective study to isolate risk factors of MSDs.

Fig. 1 .

 1 Fig. 1. Photos of sewing cycle phases. Approach phase: participants placed the needle against the back of the tissue in the right place to introduce it precisely. Needle-tissue contact phase: participants introduced and pushed the needle through the tissue and grip again the needle (to 22.4%-70.1% of the cycle). Pull thread phase: participants pulled it to stretch the sewing thread.

Fig. 2 .

 2 Fig. 2. A. Location of the 52 markers (the marker of the cervical n • 7 is not visible). b. Location of the 24 markers of the right hand (dominant). The third finger of the non-dominant hand (left) is not equipped.

Fig. 4 .

 4 Fig. 4. Time-varying joint angle for the five Degree of Freedom (DoF), which exhibited a significant difference between groups during the sewing cycle. Statistics were performed using the Statistical parametric mapping approach.

Fig. 5 .

 5 Fig. 5. Individual wrist Adduction/Abduction motion during the sewing cycle in the three groups. Data are depicted for each participant (thin line) and the average for each population (thick lines). The grey area corresponds to the standard deviation across participants. The coefficient of multiple correlation was 0.6 (CMC) for experts and 0 for both intermediates and novices.

Fig. 6 .

 6 Fig. 6. Number of components needed to explain 90% of the accounted variance.

Fig. 7 .

 7 Fig. 7. Vectors loads (left column) and coefficients (right column) of the first principal component for each group: experts (black), intermediates (magenta) and novices (blue).

Fig. 8 .

 8 Fig. 8. "Corridors" of movement, which seem to be essential for expert movement. They are compared to intermediate and novice movements. The line represents mean movement, dashed lines represent standard deviations of movement for each group. Significant differences are represented by thick line for joint movement (Statistical parametric mapping) and by asterisk for range of motion (*p < 0.5; **p < 0.1).

Table 1

 1 Joint ranges of motion of 20 DoF Labels (abbreviations Appendix 3).

	DoF Labels	Experts Range of motion ( • )	Intermediates Rangeof motion ( • )	Novices Range of motion ( • )	Main effect of Group
	Shoulder-Add/Abd	3.7	±1.7	6.2	±6.2	3.3	±7.0	ns
	Shoulder-Flex/Ext	6.2	±4.3 *	8.1	±3.8	12.6	±7.8 *	0.049
	Shoulder-Pron/Sup	18.8	±8.8	23.3	±23.8	19.2	±8.9	ns
	Elbow-Flex/Ext	20.6	±13.4	18.2	±10.6	14.5	±7.3	ns
	Wirst-Add/Abd	13.4	±4.8 *	12.8	±3.8 #	6.8	±2.4 *#	0.001
	Wirst-Flex/Ext	20.4	±8.7	20.5	±8.3	17.3	±8.9	ns
	Wrist-Pron/Sup	59.8	±20.7 *	52.4	±16.9	40.0	±14.5 *	0.037
	First-MCPAdd/Abd	14.7	±4.5	13.9	±5.7	9.9	±4.4	ns
	First-MCP-Flex/Ext	28.1	±8.5	30.9	±14.0 #	18.1	±8.4 #	0.024
	First-PIP-Flex/Ext	26.7	±6.9	32.1	±8.6	18.8	±7.8	ns
	First-DIP-Flex/Ext	20.4	±5.5	23.3	±6.6	19.5	±9.3	ns
	Middle-MCP-Add/Abd	7.0	±2	6.9	±1.3	6.3	±2.9	ns
	Middle-MCP-Flex/Ext	25.8	±5.1 *	20.6	±9.5	14.0	±4.7 *	0.006
	Middle-PIP-Flex/Ext	26.8	±7.8	26.8	±9.7	19.0	±9.1	ns
	Middle-DIP-Flex/Ext	23.5	±7.7	35.0	±17.1	22.8	±10.9	ns
	Thumb-TMC-Add/Abd	10.4	±3.9	12.8	±4.4	7.3	±2.7	ns
	Thumb-TMC-Flex/Ext	13.0	±6.4	10.7	±6.2	9.5	±5.1	ns
	Thumb-MCP-Add/Abd	9.1	±3.9	9.9	±3.1	7.4	±3.1	ns
	Thumb-MCP-Flex/Ext	13.1	±4.1	17.2	±5.9	12.7	±6.3	ns
	Thumb-IP-Flex/Ext	19.2	±6.3	21.5	±5.8	17.6	±7.7	ns

Table 2

 2 Coefficient of multiple correlation for principal components coefficients within groups.

		PC1	PC2	PC3
	Experts	0.93	0.75	0.52
	Intermediates	0.88	0.50	NA
	Novices	0.70	0.51	NA
	Principal Component (PC).

Acknowledgement

This study was funded by Institut Français du Textile et de l'Habillement (IFTH) and by l'Association Nationale Recherche Technologie (ANRT). The authors are grateful to the subjects for having accepted to participate in this study and to Aurélie Sarcher for her technical support and her knowledge on statistics.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix.1. : Type of stitch sewing used for the experimental task: the "Whip stitch" Appendix.2. : Markers dispositions "C7" was placed on cervical n • 7, "ST" on the highest part of sternum and "STX" on xiphoid process. For right side (dominant): "RSH" was placed on lateral part of acromion. "RU1","RU2", "RU3" was a cluster of markers placed on the lateral face of the arm. "REM" and "REL" was placed on medial and lateral elbow epicondyle respectively. "RU" and "RR" was placed on the extern face of ulnar and radius 5 cm proximally from the wrist and "RWU" and "RWR" on ulnar and radius distal head respectively.

For right Hand (dominant): "RC1", "RC2" and "RC3" was placed on the proximal head of 1st, 2nd, and 3rd metacarpal. "RMT" and "RPT" was placed on a rigid body on thumb metacarpal and first phalange of the thumb (to observe thumb rotation). "RM1", "RM2" and "RM3" was placed on the distal head of the 1st, 2nd , and 3rd metacarpal. "RPP1", "RPP2" and "RPP3" was placed on the proximal inter-phalangeal joints of the 1st, 2nd , and 3rd fingers. "RPM2" and "RPM3" was placed on the distal inter-phalangeal joints of the 2nd and 3rd fingers. "RPD1", "RPD2" and "RPD3" was placed on the distal head of the distal phalange.

For left side (non-dominant): "LSH" was placed on the lateral part of the acromion. "LU1","LU2", "LU3" was a cluster of markers placed on the lateral face of the arm. "LEM" and "LEL" was placed on medial and lateral elbow epicondyle respectively. "LU" and "LR" was placed on the extern face of ulnar and radius 5 cm proximally from the wrist and "LWU" and "LWR" on ulnar and radius distal head respectively.

For left hand (non-dominant): "LC1", "LC2" and "LC3" was placed on the proximal head of 1st, 2nd , and 3rd metacarpal. "LMT" and "LPT" was placed on a rigid body on metacarpal and first phalange of the thumb (to observe thumb rotation). "LM1", "LM2" and "LM3" was placed on the distal head of the 1st, 2nd , and 3rd metacarpal. "LPP1" and "LPP2" and was placed on the proximal inter-phalangeal joints of the 1 st and 2nd fingers. "RPM2" was placed on the second inter-phalangeal joints of the 2nd finger. "RPD1" and "RPD2" was placed on the distal head of the distal phalange.

Appendix.3. : Joint and movement label