Mathieu Stouf

Guillaume Witz

Jay Fisher

Nancy Kleckner

Nancy Kleckner 4d Dynamic

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Summary.

Recent studies reveal the bacterial nucleoid to be a discrete mechanically coherent object with a de ned shape and organization. Escherichia coli studies identify two global nucleoid dynamic behaviors and show that communication can occur throughout the nucleoid by transmission of information through the nucleoid matrix, with events at one position a ecting other positions far away. From this new perspective, we have investigated the roles of intra-nucleoid communication for basic aspects of chromosome function including, initiation of DNA replication, interactions with the cell membrane, and sister chromosome segregation. Using high throughput, rapid 4D time-lapse imaging and computational tools developed in the Kleckner laboratory, we have de ne the precise dynamics of oriC; replication initiation and replication fork lagging strands, in relation to one another and to other events. The nucleoid elongates in 10 minutes pulses at de ned 20 minutes interval during the cell cycle Four contraction/expansion cycles have been mesured (T1 to T4).

Introduction

For each minute in the cell cycle, the sliding window average (blue line) and the corresponding standard deviation (grey shadow) are shown.

A system for 4D analysis of nucleoid shape, organization and dynamics, with concomitant analysis of individual uorescently-tagged loci, in living cells through the cell cycle with subsequent improvements in both imaging and data analysis.

It is now possible to capture 3D images, for a hundred or more cells simultaneously, once every 30-60 sec, for multiple cell generations, with very limited photobleaching and phototoxicity.

-Take home messages

Expansion / contraction cycles are present during the all cell cycle

Arrows indicate periods of global nucleoid expansion/contraction cycles. T1-T4 were described previously (Fisher et al., 2013). Recent studies now show that these cycles occur throughout the cell cycle, not just during the time of DNA replication.Additional cycles are indicated as T(-1) and T(-2).

An unexpected behavior for the origin

After replication of the origin, sister origins remain together and split at the T1 transition (Fisher et al., 2013). The sister origins then become colocalized again and then split a second time at T2. This convergence of the origin right before T2 is an unexpected behavior and occurs in 70% of the cells observed.

Convergence and re-separation highlighted with box and nucleoid images at right (origin marked in red).

To understand and analyze the behaviors of the nucleoid along with the cell cycle, we have analysed cells with the nucleoid tagged with an hupA::mCherry construct, and a locus tagged near the origin (asnA::tetO, TetR-TFP/mVenus, 40kb from oriC), in slow growth conditions as described before (Bates and Kleckner 2005). For each color a stack of 13 z-steps has been taken every minute.

Legend

Parameters relating to origin focus (foci) and/or the nucleoid (length or local intensity) de ned as a function of time through an single entire cell cycle.

The right pannel represent the nucleoid in 3D along with the origin foci (purple) between 60 and 90 minutes.

Rapid longitudinal density waves are present The oriC foci possess a speci c dynamic (a described in 1): after cohesion the foci split and converge again before splitting a last time. The interfocal distance after the last spliting increase compared to the convergence. The replication is initiated and a focus for SSB appear and colocalyze with the origin (1, 2 and 3). Then the replisomes split and the origins are in cohesion (4). The strength behing the spliting of the SSB foci depends mostly on the replication forks. At some point, the origins split (green arrow, 5) and the replication fork converge. After, the origins converge and the replication forks can split again (6). Finally, the origins split a last time and the interfocal distance increase, along with the spliting of the SSB foci.

-Snap-shot analysis and details

-Hypothesis for the dynamic between the origin and the replication forks

Such interplays between the origins and replisome could ensure separateness not only of sisters but of Lelf/Right replichores and regularize their dispositions within the cell. We have developped a system for 4D analysis of nucleoid shape, organization and dynamics, with concomitant analysis of individual uorescently-tagged loci, in living cells through the cell cycle with subsequent improvements in both imaging and data analysis.

A speci c and concerved convergence of the origin have been observed along with a speci c temporally correlated dynamic of the SSB/replisome foci.

-Next steps

-Further explore the basis for, and signi cance of correlated origin/replisome dynamics.

-Investigate the temporal and functional relationships between nucleoid/divisome association/dissociation and replication initiation.

-Develop an optogenetic system for real-time manipulation of linkages within and between nucleoids and between the nucleoid and other cellular components.

For every two minutes, a snap shot have been taken. The signal of SSB is really noisy, but foci can still be observed.

SSB foci are splited, the origins are colocalized ( ). At the next time points, the origins split and SSB foci converge ( ). The origins stay splited but the interfocal distance is small while the replisomes split and converge twice. ( ) The origins converge a second and last time before spliting again: at this point (after 60 minutes) the interfocal distance inscrease for both SSB and origins (signi cantly) spots.

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  Nucleoid contraction/expansion cycles and longitudinal density waves 4D Dynamic of the nucleoid of Escherichia coli Stouf Mathieu, Witz Guillaume, Fisher Jay, Kleckner Nancy. MCB, Harvard Univesity, Cambridge, USA 2 -A spatio-temporal coordination of events between the origin and the replication forks 1 -A new and automatic analysis for the nucleoid dynamic 2.1 -Longitudinal kymographs showing speci c events A powerful set of home-made scripts to detect cells shape, nucleoid and foci over time and in 3D 1.2 -New insights for the origin and nucleoid dynamics The nucleoid possess an helipsoïdal structure There is a precise dynamic of the nucleoid along the cell cycle Several Z-steps acquisition of the nucleoid allow a 3D reconstruction of its shape. (1) Z-steps of the nucleoid (hupA::m-Cherry). (2-3) 3D representations of a deconvoluted nucleoid and with the origin.Progressive helix-like shape of the nucleoid of a single cell along time (every 10 minutes). Several steps are descibed: T1 and T2 are concomitent with the origin dynamic (red spots).

*

  These kymographs show the movement of the foci along the long axis of the cell, over time.The intensity for hupA::mCherry (proxy for nucleoid density) varies along the cell cycle and along the length of the cell. SSB signal appears to be correlated with low density regions of the nucleoid. The inverse is true for the origin signal.SSB foci are very mobile and converge and split several times along the cell cycle, before disappearing (105 minutes, middle kymograph). Replication is re-initiated 30 minutes before cell division.

  mVenus asnA::tetO


Stouf Mathieu

A primordial mechanism to govern nucleoid organization?

II.3 -T2 transition event stabilize the nucleoid organization

Take Home Message Methods Summary. Recent studies reveal the bacterial nucleoid to be a discrete mechanically coherent object with a de ned shape and organization. Escherichia coli studies identify two global nucleoid dynamic behaviors and show that communication can occur throughout the nucleoid by transmission of information through the nucleoid matrix, with events at one position a ecting other positions far away. From this new perspective, we have investigated the roles of intra-nucleoid communication for basic aspects of chromosome function. Using high throughput, 4D time-lapse imaging and computational tools developed in the Kleckner laboratory, we de ne the precise dynamics of oriC; nucleoid dynamic and cell septation, in relation to one another and to other events.

1) The nucleoid Expand and Compact with a 20 minutes pediodicity in slow growth condition 2) Assynchonous spiting of the origins (T2 transition) is correlated to an EC peak How the ECs cycles and the nucleoid organization correlate with each other?

Background

CM735 derivated strains with the nucleoid (hupA::mCherry) and the origin (asnA::tetO, TetR-TFP/mVenus, 40kb from oriC) tagged, were grown in di erent media: Fast: M9 Glucose CSA 30c, Slow: M9 Alanine 3aa 30c, Very Slow: M9 Alanine 3aa 21c).

-Cells were grown in grooves (Left and Right) with images taken every 30 secondes (Fast and Slow growth conditions) and 60 secondes (Very Slow growth condition).

-Images acquisition was done using Home-Made scripts and 11 z-steps were taken.

-Nucleoid uorescent images were deconvolved using Matab deconvlucy function.

-After Z projections, nucleoid and cell movies were processed throught OUFTI to extract meshes coordinates. Cell Cycle duration (min)

Step 1: Nuc. org. stable

Step 2: Septation Start

Step 3: Depth Valley inc. -Appart in fast growth, cell septation occurs in the 20% of the cell cycle.

-In all growth and 73,7% of the cases, the septation process is correlated with a compaction of the nucleoid. Cell Birth Cell Div.

  Green / Red dots = position of the 2nd and 3rd deepest valley (collored by their position) (B) Red curve = Depth of the deepest valley (C) White curve = Nucleoid Complexity = Fluctuations of the sum of the depth of the three deepest valleys. Green curve = Raw -Fit of the nucleoid Length (D) Relative timings of T1 and T2 transitions (E) Examples of T1 and T2 transitions from the origin dynamics II.2 Duality in all growth conditions II -The T2 transition, a check-point in the organization and behavior of the nucleoid II.4 -Septation does not a ect the nucleoid is organized at later events. -From fast to slow growing cells, the nucleoid is more and more organized. -The sub-units of the nucleoid increase is length from birth to division. stage, the localisation of the deepest valley is always localized close tothe center of the nucleoid. -The deepest valley can localize at the center of the nucleoid quite early. -The deepest valley stabilise at the center before the duality is visible. -The depth of the deepest valley increase in the late 20% of the cell cycle FAST VERY SLOW SLOW Time (frame) Position of the deepest valley Depth of the deepest valley Time (frame) Position of the deepest valley Depth of the deepest valley 1) ZY projection for each from using the nucleoid or cell meshes. 2) Detection of the Valleys from Intensity plot 3) Comparison of the timing ot T2 transitions or other events with the changes in numbers and depths of the valleys is correlated with stabilization of the deepest valley at the nucleoid center (87.5%) -Stabilization of deepest valley is followed by apperance of smaller valleys (1/4 -3/4), early in fast growth, late in slower growth. -Fluctuations in Length (Green Curve) and Complexity (White Curve) are correlated during most of the nucleoid/Green / Red dots = position of the 2nd and 3rd deepest valley (collored by their position) (B) Red curve = Depth of the deepest valley. Magenta = Cell Width at the center. (C) Red curve = Depth of the deepest valley. Magenta = Cell Width at the center. Green curve = Raw -Fit of the nucleoid Length (D) Relative timings from the reaching state of a stable nucleoid (black) to the begining of the septation process (Green) and last increase in depth for the deepest valley (Blue).

  the begining of cell septation ( and last increase in depth of the deepest valley. These two events are separated by less than 2 minutes (1% relative time).

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  Relative time between Step 1 and Step 2-3 is constant in all growth and Using the lowest intensities (Valleys) to de ne the organization of the