Minimal requirements for robust cell size control in eukaryotic cells

Most cell types living in a stable environment tend to keep a constant characteristic size over successive generations. Size homeostasis requires that cells exert a tight control over the size at which they divide. Cell size control is not only robust against various noises, but also highly flexible since cell sizes can vary tremendously, notably as a function of nutrient levels. We formulated a minimal mathematical model of the eukaryotic cell cycle in which the cell size control operates through a cell growth-dependent bifurcation in the cell cycle dynamics. Such a bifurcation mechanism can readily explain the occurrence of a minimum critical size at division under limiting growth conditions. However, it also predicts that cells should become progressively larger and larger under prolific growth conditions. We argue that the cell size control can be reinforced at fast growth rates by adding a new cell cycle inhibitory activity whose strength would increase with the cell growth rate. We further show that various sources of noise may also generate a large variability in cell size at division and interdivision time that exhibit characteristic exponential tail distributions, without compromising the robustness of the cell size control.     

Linking cell division to cell growth in a spatiotemporal model of the cell cycle

Cell division must be tightly coupled to cell growth in order to maintain cell size, yet the mechanisms linking these two processes are unclear. It is known that almost all proteins involved in cell division shuttle between cytoplasm and nucleus during the cell cycle; however, the implications of this process for cell cycle dynamics and its coupling to cell growth remains to be elucidated. We developed mathematical models of the cell cycle which incorporate protein translocation between cytoplasm and nucleus. We show that protein translocation between cytoplasm and nucleus not only modulates temporal cell cycle dynamics, but also provides a natural mechanism coupling cell division to cell growth. This coupling is mediated by the effect of cytoplasmic-to-nuclear size ratio on the activation threshold of critical cell cycle proteins, leading to the size-sensing checkpoint (sizer) and the size-independent clock (timer) observed in many cell cycle experiments.     

Ssp1 promotes actin depolymerization and is involved in stress response and new end take-off control in fission yeast

The ssp1 gene encodes a protein kinase involved in alteration of cell polarity in Schizosaccharomyces pombe. ssp1 deletion causes stress sensitivity, reminiscent of defects in the stress-activated MAP kinase, Spc1; however, the two protein kinases do not act through the same pathway. Ssp1 is localized mainly in the cytoplasm, but after a rise in external osmolarity it is rapidly recruited to the plasma membrane, preferentially to active growth zones and septa. Loss of Ssp1 function inhibits actin relocalization during osmotic stress, in cdc3 and cdc8 mutant backgrounds, and in the presence of latrunculin A, implicating Ssp1 in promotion of actin depolymerization. We propose a model in which Ssp1 can be activated independently of Spc1 and can partially compensate for its loss. The ssp1 deletion mutant exhibited monopolar actin distribution, but new end take-off (NETO) could be induced in these cells by exposure to KCl or to latrunculin A pulse treatment. This treatment induced NETO in cdc10 cells arrested in G1 but not in tea1 cells. This suggests that cells that contain intact cell end markers are competent to undergo NETO throughout interphase, and Ssp1 is involved in generating the NETO stimulus by enlarging the actin monomer pool.     

Tea3p is a cell end marker activating polarized growth in Schizosaccharomyces pombe

Eukaryotic cells are often polarized in their cytoplasmic structures, and this can be important for their function. The fission yeast Schizosaccharomyces pombe is a highly polarized cell that extends bipolarly along a single axis to generate a rod-shaped cell. It divides by medial fission to generate two equal-sized daughter cells that resume growth only at the old end. Once these cells have reached a particular length, they undergo NETO, new end take-off, whereby growth is activated at the other end to generate bipolarly extending cells. The activation and positioning of these growth zones are essential for maintaining growth in a straight line. Genetic analyses have identified many proteins involved in this process, like the cell end markers Tea1p and Pom1p and the kinases Orb2p/Shk1p/Pak1, Ssp1p, and Wee1p. Here, we describe tea3, a gene encoding a tea1-like protein with some similarities to ERM proteins. Tea3p is required for efficient NETO and for the proper placement of the septum. Like Pom1p, Tea3p localizes to cell ends, and its localization depends on microtubules and Tea1p. We propose that Tea3p is a novel cell end marker required specifically to activate polarized cell growth at the second end during NETO.     

Modeling the Dynamics of Cdc42 Oscillation in Fission Yeast

Regulation of polarized cell growth is essential for many cellular processes, including spatial coordination of cell morphology changes during growth and division. We present a mathematical model of the core mechanism responsible for the regulation of polarized growth dynamics by the small GTPase Cdc42. The model is based on the competition of growth zones of Cdc42 localized at the cell tips for a common substrate (inactive Cdc42) that diffuses in the cytosol. We consider several potential ways of implementing negative feedback between Cd42 and its GEF in this model that would be consistent with the observed oscillations of Cdc42 in fission yeast. We analyze the bifurcations in this model as the cell length increases, and total amount of Cdc42 and GEF increase. Symmetric antiphase oscillations at two tips emerge via saddle-homoclinic bifurcations or Hopf bifurcations. We find that a stable oscillation and a stable steady state can coexist, which is consistent with the experimental finding that only 50% of bipolar cells oscillate. The mean amplitude and period can be tuned by parameters involved in the negative feedback. We link modifications in the parameters of the model to observed mutant phenotypes. Our model suggests that negative feedback is more likely to be acting through inhibition of GEF association rather than upregulation of GEF dissociation.     

New End Take Off: Regulating Cell Polarity during the Fission

Cell polarisation is a major event of the cell cycle and underlies the function of mostcells. Cell polarity is often achieved through the coordinated organisation of themicrotubule and actin cytoskeletons. Dramatic changes in cell polarisation occur duringthe cell cycle and are subject to regulation by cell cycle controls. Cells of the fission yeastSchizosaccharomyces pombe grow by tip extension in a cell cycle-controlled manner.During G2 phase, these cells exhibit a transition in cell polarisation known as New EndTake Off (NETO), in which monopolar cells initiate bipolar growth. Dynamicmicrotubules contribute to this process by depositing at cell ends the microtubule plusend proteins tea1p and tea4p, which are necessary for NETO. We discuss here how theseproteins may recruit for3p, a formin responsible for actin nucleation, as well as two otheractin binding proteins, bud6p and sla2p, to initiate cell polarisation at the new end of thecell. Thus, the study of NETO is revealing a mechanism by which the plus ends ofmicrotubules regulate the spatial organisation of actin.     

Calcineurin ensures a link between the DNA replication checkpoint and microtubule-dependent polarized growth

Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (+TIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signalling pathway linking +TIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The +TIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis.     

Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors

Primary cilia are displayed during the G(0)/G(1) phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1(T94E) mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G(1) length, cell cycle entry and fate determination of these cells during corticogenesis.     

ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as ‘New End Take Off’ (NETO). Here I report the identification of a gene, arf6⁺, encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6Δ cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.     

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.     

Modeling cell proliferation for simulating three-dimensional tissue morphogenesis based on a reversible network reconnection framework

Tissue morphogenesis in multicellular organisms is accompanied by proliferative cell behaviors: cell division (increase in cell number after each cell cycle) and cell growth (increase in cell volume during each cell cycle). These proliferative cell behaviors can be regulated by multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze multicellular dynamics, a reversible network reconnection (RNR) model has been suggested, in which each cell shape is expressed by a single polyhedron. In this study, to apply the RNR model to simulate tissue morphogenesis involving proliferative cell behaviors, we model cell proliferation based on a RNR model framework. In this model, cell division was expressed by dividing a polyhedron at a planar surface for which cell division behaviors were characterized by three quantities: timing, intracellular position, and normal direction of the dividing plane. In addition, cell growth was expressed by volume growth as a function of individual cell times within their respective cell cycles. Numerical simulations using the proposed model showed that tissues grew during successive cell divisions with several cell cycle times. During these processes, the cell number in tissues increased while maintaining individual cell size and shape. Furthermore, tissue morphology dramatically changed based on different regulations of cell division directions. Thus, the proposed model successfully provided a basis for expressing proliferative cell behaviors during morphogenesis based on a RNR model framework.     

Long-Term Single Cell Analysis of S. pombe on a Microfluidic Microchemostat Array

Although Schyzosaccharomyces pombe is one of the principal model organisms for studying the cell cycle, surprisingly few methods have characterized S. pombe growth on the single cell level, and no methods exist capable of analyzing thousands of cells and tens of thousands of cell division events. We developed an automated microfluidic platform permitting S. pombe to be grown on-chip for several days under defined and changeable conditions. We developed an image processing pipeline to extract and quantitate several physiological parameters including cell length, time to division, and elongation rate without requiring synchronization of the culture. Over a period of 50 hours our platform analyzed over 100000 cell division events and reconstructed single cell lineages up to 10 generations in length. We characterized cell lengths and division times in a temperature shift experiment in which cells were initially grown at 30°C and transitioned to 25°C. Although cell length was identical at both temperatures at steady-state, we observed transient changes in cell length if the temperature shift took place during a critical phase of the cell cycle. We further show that cells born with normal length do divide over a wide range of cell lengths and that cell length appears to be controlled in the second generation, were large newly born cells have a tendency to divide more rapidly and thus at a normalized cell size. The platform is thus applicable to measure fine-details in cell cycle dynamics, should be a useful tool to decipher the molecular mechanism underlying size homeostasis, and will be generally applicable to study processes on the single cell level that require large numbers of precision measurements and single cell lineages.     

Cell division in Escherichia coli minB mutants

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated. The main findings were as follows. In the minB mutants, polar and non-polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re-evaluation of the role of the MinB system in the E. coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle.     

Cycling in a crowd: Coordination of plant cell division,growth, and cell fate

The reiterative organogenesis that drives plant growth relies on the constant production of new cells, which remain encased by interconnected cell walls. For these reasons, plant morphogenesis strictly depends on the rate and orientation of both cell division and cell growth. Important progress has been made in recent years in understanding how cell cycle progression and the orientation of cell divisions are coordinated with cell and organ growth and with the acquisition of specialized cell fates. We review basic concepts and players in plant cell cycle and division, and then focus on their links to growth-related cues, such as metabolic state, cell size, cell geometry, and cell mechanics, and on how cell cycle progression and cell division are linked to specific cell fates. The retinoblastoma pathway has emerged as a major player in the coordination of the cell cycle with both growth and cell identity, while microtubule dynamics are central in the coordination of oriented cell divisions. Future challenges include clarifying feedbacks between growth and cell cycle progression, revealing the molecular basis of cell division orientation in response to mechanical and chemical signals, and probing the links between cell fate changes and chromatin dynamics during the cell cycle.

Plant cell cycle and division are linked to specific cell fates and respond to growth-related cues, such as metabolic state, cell size, cell shape, and mechanical stress.     

Analysis of protein distribution in budding yeast

Flow cytometry is a fast and sensitive method that allows monitoring of different cellular parameters on large samples of a population. Protein distributons give relevant information on growth dynamics, since they are related to the age distribution and depend on the law of growth of the population and the law of protein accumulation during the cell cycle. We analyzed protein distributions to evaluate alternative growth models for the budding yeast Saccharomyces cerevisiae and to monitor the changes in population dynamics that result from environmental modifications; such an analysis could potentially give parameters useful in the control of biotechnological processes. Theoretical protein distributions (taking into account the unequal division of yeast cells and the exponential law of protein accumulation during a cell cycle) quantitatively fit experimental distributions, once appropriate variability sources are introduced. Best fits are obtained when the protein threshold required for bud emergence increases at each new generation of parent cells.