Computational Modeling of 3D Actin Organization through Polymerization, Turnover, Crosslinking, and Motor Pulling

The cellular actin cytoskeleton is an intricate system of actin filaments that supports cell morphology and is crucial for numerous cell functions including cell growth and cell division. Among the most important actin cytoskeleton structures are actin cables and cytokinetic ring, which are bundles of actin filaments. The actin cables span the cell and serve as tracks for vesicle transport while the actin cytokinetic ring forms in the middle and constricts to divide the cell. The focus of my work is to gain a quantitative understanding of how such cables and ring are formed, what are the essential components, how overexpression and underexpression of certain proteins will affect the structure and dynamics. I built a 3D computational model that starts out from the basic physical and mechanical properties of actin filaments and accounts for known interactions with other proteins, to reproduce the experimental observations of the actin cytoskeleton in different cell systems and further make testable predictions for cell mutants.First, I modeled individual actin filament as a semiflexible worm like chain. I coarse grained the filamentous actin segment using a bead-spring model with spring, bending and thermal forces. This model represents of the actin filament\u27s spatial and dynamical properties. I tested that the model reproduces the correct persistence length, relaxation dynamics and equipartition of energy.Second, to obtain a quantitative understanding of these actin structures and dynamics in fission yeast, I extended the individual actin filament model and added actin-interacting factors. Polymerization out of formin cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin, are simulated as growth, cross-linking, pulling and turnover of the semiflexible polymers. With the above mechanisms my quantitative model generated actin cable structures and dynamics similar to those observed in live cell experiments. The simulations reproduced the particular actin cable structures in myoVΔ cells and predicted the effect of increased myosin V pulling. I found that increasing cross-linking parameters generated thicker actin cables and led to anti-parallel and parallel phases with straight or curved cables. I further analyzed the cable number, curvature and loop occurrences of experimental images of cells overexpressing crosslinkers and cell treated with drugs that depolymerize actin patches, provided by our collaborator Damien Laporte. Our predictions are in quantitative agreement with the experiments. Furthermore, the model predicts that clustering of formins at cell tips promotes actin cable formation.Third, I adapted the actin cable model to budding yeast, another well-studied model organism. Budding yeast differs from fission yeast in that it has a more complex geometry and different types of interacting proteins. I refined the previous fission yeast actin cable model by considering a more accurate model of orientation-dependent crosslinking by fimbrin and a more accurate aging mechanism for turnover. It also included polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosins stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which I suggest as a model for the assembly of actin filaments to the contractile ring.Finally, my colleague Dr. Tamara Bidone and I further extended the model to simulate the actin contractile ring. We showed that the ring formation region in parameter space lies close to regions leading to clumps, meshworks or double rings, and stars/cables, which are consistent with prior experiments with mutations that alter the morphology of the condensing network. We also quantified tension along actin filaments and forces on nodes during ring assembly and showed that the mechanisms describing ring assembly can also drive ring constriction once the ring is formed.In summary, this work provides a numerical way to study the morphology and dynamics of the actin cytoskeleton in model cell organisms. Combining simulated, analytical and experimental results, the proposed model with minimal set of interactions successfully reproduced experimental observations and made predictions for further studies

Anaphase B.

Anaphase B spindle elongation is characterized by the sliding apart of overlapping antiparallel interpolar (ip) microtubules (MTs) as the two opposite spindle poles separate, pulling along disjoined sister chromatids, thereby contributing to chromosome segregation and the propagation of all cellular life. The major biochemical "modules" that cooperate to mediate pole-pole separation include: (i) midzone pushing or (ii) braking by MT crosslinkers, such as kinesin-5 motors, which facilitate or restrict the outward sliding of antiparallel interpolar MTs (ipMTs); (iii) cortical pulling by disassembling astral MTs (aMTs) and/or dynein motors that pull aMTs outwards; (iv) ipMT plus end dynamics, notably net polymerization; and (v) ipMT minus end depolymerization manifest as poleward flux. The differential combination of these modules in different cell types produces diversity in the anaphase B mechanism. Combinations of antagonist modules can create a force balance that maintains the dynamic pre-anaphase B spindle at constant length. Tipping such a force balance at anaphase B onset can initiate and control the rate of spindle elongation. The activities of the basic motor filament components of the anaphase B machinery are controlled by a network of non-motor MT-associated proteins (MAPs), for example the key MT cross-linker, Ase1p/PRC1, and various cell-cycle kinases, phosphatases, and proteases. This review focuses on the molecular mechanisms of anaphase B spindle elongation in eukaryotic cells and briefly mentions bacterial DNA segregation systems that operate by spindle elongation

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in eLife 5 (2016): e14216, doi:10.7554/eLife.14216.RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.Work in FV’s laboratory is supported by the National Institutes of Health R01 grant number GM095867. Part of this work was also supported by NSF grant 0745129. TT was supported by Japan Society for the Promotion of Science grants 16H02503 and 16K14672 and by Cancer Research UK

Physical Models of Cell Polarity

Fission yeast is a pill-shaped unicellular organism, and before dividing it grows by extension at the tips to double the original length. This work consists of mathematical models for how fission yeast controls this growth process. The models presented are either developed in collaboration with experimentalists or using published experimental work on this organism.First, in collaboration with experimentalists Maitreyi Das and Fulvia Verde, we examine the organization of the signaling protein Cdc42, which we implicate as a central part of a control system for polarized growth. Cdc42, a member of the Rho family of proteins, binds to the inner membrane of the cell tips where growth occurs. In collaboration, we find that the fraction of Cdc42 bound to a given cell tip correlates to its growth rate, and that the amount of bound Cdc42 undergoes anti-correlated oscillations between the cell tips. We present a model that describes how Cdc42 and related proteins effect this organization, and shows how the oscillations could function as an exploratory mechanism to help the system overcome a kinetic barrier. Experimental results from our collaborators, such as a loss of correlation in very long cells and a reorganization after disruptive drug treatment, validate the model.Next, using experimental results from literature, we turn to the patterned remodeling of the cell wall. We make a hypothesis that extends the result that Cdc42 marks cell tips for growth from previous work: that Cdc42 marks sites for growth on a microscopic level. A model for the fission yeast cell as an elastic shell being remodeled under turgor pressure at a rate that depends on cortical Cdc42 levels reproduces essential experimental results, namely the ratio of signal width to cell diameter and a linear relation between growth rate and pressure, and gives an estimation of the wall remodeling rate at the cell tips. Since this model predicts that cell diameter depends crucially on the width of a Cdc42 signal, we consider the plausibility of mechanisms for establishing the width of that signal. We find that stronger-than-linear feedback from cell diameter to signal width leads to unstable width regulation, and propose an independent length scale such as from a reaction-diffusion-type mechanism for a cell-diameter-independent Cdc42 signal width. Finally, we describe a mathematical model consisting of Cdc42-signal-dependent cell growth, diffusing Cdc42 growth zones with native width, and an axis-sensing microtubule-based system capable of delivering landmark proteins to the cell tips that bias the diffusion of the growth zones. Parameter dependence of the model is explored, and we show that such a model can give straight, bent, and wide cells, all of which have been observed by experimentalists. We argue that such a model is consistent with the roles of cytoskeleton- and signal-related proteins and known aberrant shapes of mutant cells.As a whole, this work provides mechanistic insight into the system regulating shape and growth in one important model organism

Cytoskeleton and Cell Motility

The present article is an invited contribution to the Encyclopedia of Complexity and System Science, Robert A. Meyers Ed., Springer New York (2009). It is a review of the biophysical mechanisms that underly cell motility. It mainly focuses on the eukaryotic cytoskeleton and cell-motility mechanisms. Bacterial motility as well as the composition of the prokaryotic cytoskeleton is only briefly mentioned. The article is organized as follows. In Section III, I first present an overview of the diversity of cellular motility mechanisms, which might at first glance be categorized into two different types of behaviors, namely "swimming" and "crawling". Intracellular transport, mitosis - or cell division - as well as other extensions of cell motility that rely on the same essential machinery are briefly sketched. In Section IV, I introduce the molecular machinery that underlies cell motility - the cytoskeleton - as well as its interactions with the external environment of the cell and its main regulatory pathways. Sections IV D to IV F are more detailed in their biochemical presentations; readers primarily interested in the theoretical modeling of cell motility might want to skip these sections in a first reading. I then describe the motility mechanisms that rely essentially on polymerization-depolymerization dynamics of cytoskeleton filaments in Section V, and the ones that rely essentially on the activity of motor proteins in Section VI. Finally, Section VII is devoted to the description of the integrated approaches that have been developed recently to try to understand the cooperative phenomena that underly self-organization of the cell cytoskeleton as a whole.Comment: 31 pages, 16 figures, 295 reference

Heterologous expression of the mammalian microtubule associated proteins (MAPs) tau, MAP2c and MAP4 in the fission yeast, Schizosaccharomyces pombe

The mammalian microtubule associated proteins (MAPs) tau, MAP2c and MAP4 were subcloned and expressed in the fission yeast Schizosaccharomyces pombe using the thiamine repressible pREP1 vector. Tau, MAP2c and MAP4 have similar C-terminal microtubule binding domains, but unique N-terminal projection domains. At the start of this study, there were no known MAPs in S. pombe and therefore this appeared to be a unique in vivo system to both study the roles of the three mammalian MAPs and to further define the function of microtubules. All three MAPs inhibited growth of wild type cells at 36°C over a range of temperatures. However each MAP produced distinct phenotypes in fission yeast, indicating that their effect was specific for that MAP. Tau expression resulted in a weak phenotype of long, multiseptate or branched cells. The MAP2c-induced phenotype was stronger, and resulted in long cells with bulbous ends, whilst MAP4 expression produced bent or hammer shaped cells. The MAPs tau and MAP2c accelerated and slowed entry into mitosis, respectively, of G2-arrested cdc25.211 cells. Expression of tau and MAP2c in tea1 cells (which plays a role in directing the cell to grow along a perfectly opposed longitudinal axis) and tea2 cells (which codes for a kinesin) result in distorted shapes and loss of the original phenotype. Immunofluorescence studies showed that each MAP had a unique effect not only on the cell phenotype but also on microtubule organisation of the cell. Tau caused microtubule bundling and displacement towards the cell periphery, MAP2c resulted in short microtubules around the nucleus, whilst MAP4 caused total depolymerisation of interphase microtubules. Both tau and MAP2c appeared to bind to microtubules, but MAP4 was seen distributed throughout the cell. Similarly, MAP2c caused the formation of short microtubules in teal cells, but tau had very little effect. tea2 control cells have short microtubules, and tau expression resulted in even shorter microtubules near the nucleus. MAP2c expression in tea2, however, resulted in microtubule depolymerisation, and assymetrical distribution towards one end of the cell. Tau and MAP2c also appeared to rescue the combined sensitivity of fission yeast to the cold and the microtubule depolymerisation agent, thiabendazole (TBZ). This observation was used as a strategy to isolate possible S. pombe MAPs by expressing an S. pombe cDNA library subcloned in pREP. Eight clones were isolated, and sequence analysis of two of those clones revealed that they coded for fatty acid synthetase (lsd1+) and the ribosomal protein L19

Dynamique de la paroi cellulaire dans la régulation de la morphogenèse et de la croissance cellulaire

Cells in nature develop in a wide range of forms, following diverse growth patterns. Despite the importance of these fundamental processes, how cells regulate their growth and morphogenesis is still poorly understood. In this thesis, I explored these processes, focusing my investigations on tip growing walled cells and in particular, by exploiting the fission yeast Schyzosaccharomyces pombe, adopting a mainly biomechanical approach. To this aim, I first developed novel methods to measure key cell wall mechanical parameters in vivo and in large scale, which allowed the very first observations of cell wall dynamics. This revealed that the cell wall is softer and highly variable at growing poles, and almost stable and stiffer at non-growing sites. During elongation, there is an interplay between wall mechanics and cell growth, whose active control allows cell expansion while preserving cell integrity. In addition, I observed that there is a strong correlation between cell wall mechanics and cell morphology, and ectopic perturbations of wall properties directly affect shape establishment and maintenance. Together my results show that the regulation of wall mechanics is fundamental in the determination of cell dynamics in tip growing walled cells. Moreover, this suggests that dynamic observation of cell surface mechanics is crucial for a complete understanding of multifactorial and complex processes as growth and morphogenesis.Les cellules dans la nature se développent dans un large éventail de formes, suivant divers modèles de croissance. Malgré l'importance de ces processus fondamentaux, la façon dont les cellules régulent leur croissance et leur morphogenèse est encore mal comprise. Dans cette thèse, j'ai exploré ces aspects, avec une approche principalement biomécanique, en concentrant mes investigations sur des cellules à paroi à croissance de pointe et en exploitant en particulier la levure fissipare Schyzosaccharomyces pombe. J'ai d'abord développé de nouvelles méthodes pour mesurer les paramètres mécaniques clés de la paroi cellulaire in vivo et à grande échelle, ce qui a permis les premières observations de la dynamique des parois cellulaires. Ceci a révélé que la paroi cellulaire est plus souple et très variable au niveau des pôles de croissance, et presque stable et plus rigide dans les sites non cultivés. Au cours de l'allongement, il existe une interaction entre la mécanique des parois et la croissance cellulaire, dont le contrôle actif permet l'expansion cellulaire tout en préservant l'intégrité des cellules. De plus, j'ai observé qu'il existe une forte corrélation entre la mécanique des parois cellulaires et la morphologie cellulaire, et des perturbations des propriétés de la paroi affectent directement l'établissement et la maintenance de la forme. Ensemble, mes résultats montrent que la régulation de la paroi est fondamentale dans la détermination de la dynamique cellulaire dans les cellules à parois épaissies. Globalement, cela suggère que l'observation dynamique de la mécanique de surface cellulaire est essentielle pour une compréhension complète des processus multifactoriels et complexes comme la croissance et la morphogenèse