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In nature, several ciliated protists possess the remarkable ability to execute ultrafast motions using protein assemblies called myonemes, which contract in response to Ca2+ions. Existing theories, such as actomyosin contractility and macroscopic biomechanical latches, do not adequately describe these systems, necessitating new models to understand their mechanisms. In this study, we image and quantitatively analyze the contractile kinematics observed in two ciliated protists (Vorticella spandSpirostomum sp), and, based on the mechanochemistry of these organisms, we propose a minimal mathematical model that reproduces our observations as well as those published previously. Analyzing the model reveals three distinct dynamic regimes, differentiated by the rate of chemical driving and the importance of inertia. We characterize their unique scaling behaviors and kinematic signatures. Besides providing insights into Ca2+-powered myoneme contraction in protists, our work may also inform the rational design of ultrafast bioengineered systems such as active synthetic cells.

The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeastSchizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak non-ergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, cytoskeletal effects, cell cycle stage and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not “well-mixed” but represents a highly heterogeneous environment in which subcellular components at the 40-nm sizescale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.SignificanceBiophysical properties of the cytoplasm influence many cellular processes, from differentiation to cytoskeletal dynamics, yet little is known about how tightly cells control these properties. We developed a combined experimental and computational approach to analyze cytoplasmic heterogeneity through the lens of diffusion. We find that the apparent cytoplasmic viscosity varies tremendously – over 100-fold within any individual cell, and over 10-fold among individual cells when comparing averages of all particles measured for each cell. The variance was largely independent of temperature, the cytoskeleton, cell cycle stage, and localization, but was magnified under hyperosmotic shock. This suggests that cytoplasmic heterogeneity contributes substantially to biological variability within and between cells, and has significant implications for any cellular process that depends on diffusion.Graphical abstract

The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules and an increase in crowding in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.

The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeast and in mammalian cell lines. We found differences in particle motility between the nucleus and the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm.

Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density in fission yeast is maintained in a narrow distribution and exhibits homeostatic behavior. However, density varied during the cell cycle, decreasing during G2, increasing in mitosis and cytokinesis, and dropping rapidly at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages exacerbated density changes. Spatially heterogeneous patterns of density suggested links between density regulation, tip growth, and intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.

Fundamental mechanisms governing cell size control and homeostasis are still poorly understood. The relationship between sizes at division and birth in single cells is used as a metric to categorize the basis of size homeostasis [1–3]. Cells dividing at a fixed size regardless of birth size (sizer) are expected to show a division-birth slope of 0, whereas cells dividing after growing for a fixed size increment (adder) have an expected slope of +1 [4]. These two theoretical values are, however, rarely experimentally observed. For example, rod-shaped fission yeast Schizosaccharomyces pombe cells, which divide at a fixed surface area [5, 6], exhibit a division-birth slope for cell lengths of 0.25±0.02, significantly different from the expected sizer value of zero. Here we investigate possible reasons for this discrepancy by developing a mathematical model of sizer control including the relevant sources of variation. Our results support pure sizer control and show that deviation from zero slope is exaggerated by measurement of an inappropriate geometrical quantity (e.g., length instead of area), combined with cell-to-cell radius variability. The model predicts that mutants with greater errors in size sensing or septum positioning paradoxically appear to behave as better sizers. Furthermore, accounting for cell width variability, we show that pure sizer control can in some circumstances reproduce the apparent adder behaviour observed in E. coli. These findings demonstrate that analysis of geometric variation can lead to new insights into cell size control. SIGNIFICANCE How cells control their size is a fundamental but still open question. It has been assumed that the two principle methods of control, namely sizer (where cells always divide at a fixed size) and adder (where cells always add a fixed size increment), can be identified because the correlation between size at birth and size at division is equal to 0 and to +1, respectively. However, experiments mainly provide numbers in between these two values. We show that use of cell length as the size measure, together with cell-to-cell radius variability, can explain this discrepancy and support pure sizer control in fission yeast. Surprisingly, the same phenomenon might exclude adder control in bacteria like E. coli.

International audience; The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli’s inner membrane from the cell wall. Shrinkage was accompanied by a ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon-source quality. Robust recovery from starvation required the Tol-Pal system, highlighting the importance of envelope coupling during recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.Significance statementBacterial cells constantly face nutrient fluctuations in their natural environments. While previous studies have identified gene expression changes upon nutrient depletion, it is much less well known how cellular morphology and cytoplasmic properties respond to shifts in nutrient availability. Here, we discovered that switching fast-growing Escherichia coli cells to nutrient-free conditions results in substantial shrinkage of the inner membrane away from the cell wall, especially at the new pole. Shrinkage was primarily driven by loss of cytoplasmic water contents. Shrinkage was also exhibited by cells naturally entering stationary phase, highlighting its biological relevance across starvation conditions. The membrane-spanning Tol-Pal system was critical for robust entry into and recovery from shrinkage, indicating the importance of cell-envelope homeostasis in surviving nutrient starvation.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functionsin vivoremain poorly understood. Here, we probed the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that both the rates of microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, while a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowdingper se. We reconstituted these effects on microtubulesin vitroby tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of cytoplasm modulates the reactions underlying microtubule dynamic behaviors.

Intracellular density is a critical parameter that impacts the physical nature of the cytoplasm and has the potential to globally affect cellular processes. How density is regulated during cell growth is poorly understood. Here, using a new quantitative phase imaging method, we show that dry-mass density varies during the cell cycle in the fission yeast Schizosaccharomyces pombe. Density decreased during G2 phase, increased in mitosis and cytokinesis, and rapidly dropped at cell birth. These density variations can be explained mostly by a constant rate of biomass synthesis throughout the cell cycle, coupled to slowdown of volume growth during mitosis and cytokinesis and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages led to continued increases or decreases in density. Spatially heterogeneous patterns of density suggested a link between tip growth and density regulation. Density differences between daughter cells in cells delayed in cytokinesis resulted in bending of the septum away from the high-density daughter, suggesting that intracellular density correlates with turgor pressure. Our results demonstrate that the systematic variations in density during the cell cycle are determined predominantly by modulation of volume expansion, and reveal functional consequences of intracellular density gradients.

Cell size is partly determined through coordination between cell growth and division. How this coordination is achieved mechanistically remains mostly unknown. However, quantitative experiments together with computational modelling have reinvigorated the field and are elucidating underlying molecular processes. Size homeostasis may be achieved through different modes of regulation, including “sizers”, “adders” and “timers.” For sizer regulation, the cell division cycle does not proceed until a minimal size has been reached, requiring that the cell monitors its own size. Here, we highlight progress in defining sizer mechanisms in fission and budding yeasts showing how accumulation or dilution of key molecules can be used to monitor cell size during growth. We also discuss a potential role for sizers in bacterial size control., Highlights • Sizer mechanisms where cells sense their own size play a crucial role in size control. • Activator accumulation model proposed for a sizer mechanism in fission yeast. • Inhibitor dilution model proposed for a sizer mechanism in budding yeast. • Sizer mechanisms may play a hidden role in bacterial size control.

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

Summary How cell size is determined and maintained remains unclear, even in simple model organisms. In proliferating cells, cell size is regulated by coordinating growth and division through sizer, adder, or timer mechanisms or through some combination [1, 2]. Currently, the best-characterized example of sizer behavior is in fission yeast, Schizosaccharomyces pombe, which enters mitosis at a minimal cell size threshold. The peripheral membrane kinase Cdr2 localizes in clusters (nodes) on the medial plasma membrane and promotes mitotic entry [3]. Here, we show that the Cdr2 nodal density, which scales with cell size, is used by the cell to sense and control its size. By analyzing cells of different widths, we first show that cdr2+ cells divide at a fixed cell surface area. However, division in the cdr2Δ mutant is more closely specified by cell volume, suggesting that Cdr2 is essential for area sensing and supporting the existence of a Cdr2-independent secondary sizer mechanism more closely based on volume. To investigate how Cdr2 nodes may sense area, we derive a minimal mathematical model that incorporates the cytoplasmic kinase Ssp1 as a Cdr2 activator. The model predicts that a cdr2 mutant in an Ssp1 phosphorylation site (cdr2-T166A) [4] should form nodes whose density registers cell length. We confirm this prediction experimentally and find that thin cells now follow this new scaling by dividing at constant length instead of area. This work supports the role of Cdr2 as a sizer factor and highlights the importance of studying geometrical aspects of size control., Graphical Abstract, Highlights • Cdr2 nodal density mediates surface-area-based size control in fission yeast • Mathematical modeling predicts length scaling for nodal mutant Cdr2-T166A density • Thin cdr2-T166A cells divide using length, demonstrating size control reprogramming • A secondary sizer mechanism based more closely on volume is active in a cdr2 mutant, By using cells of different widths, Facchetti et al. show that fission yeast size homeostasis is based on cell surface area as registered by Cdr2 nodal density. A mathematical model allows reprogramming of this Cdr2-based size regulation from area to length. Secondary size control in a cdr2 mutant more closely based on volume is also uncovered.

© 2019 Cook et al., XMAP215/Stu2/Alp14 accelerates tubulin polymerization while processively tracking microtubule (MT) plus ends via tumor overexpressed gene (TOG) domain arrays. It remains poorly understood how these functions arise from tubulin recruitment, mediated by the distinct TOG1 and TOG2 domains, or the assembly of these arrays into large square complexes. Here, we describe a relationship between MT plus-end tracking and polymerase functions revealing their distinct origin within TOG arrays. We study Alp14 mutants designed based on structural models, with defects in either tubulin recruitment or self-organization. Using in vivo live imaging in fission yeast and in vitro MT dynamics assays, we show that tubulins recruited by TOG1 and TOG2 serve concerted, yet distinct, roles in MT plus-end tracking and polymerase functions. TOG1 is critical for processive plus-end tracking, whereas TOG2 is critical for accelerating tubulin polymerization. Inactivating interfaces that stabilize square complexes lead to defects in both processive MT plus-end tracking and polymerase. Our studies suggest that a dynamic cycle between square and unfurled TOG array states gives rise to processive polymerase activity at MT plus ends., B.D.C. was supported by the UC Davis Training Program in Molecular and Cellular Biology (funded in part by NIH-T32-GM007377). J.A.-B. is supported by NIH-GM110283 and NSF-1615991. I.F.-P. is supported by the research program funds from the Universidad Pablo De Olavide. F.C. is supported by NIH-GM115185.

Cell growth is a complex process in which cells synthesize cellular components while they increase in size. It is generally assumed that the rate of biosynthesis must somehow be coordinated with the rate of growth in order to maintain intracellular concentrations. However, little is known about potential feedback mechanisms that could achieve proteome homeostasis, or the consequences when this homeostasis is perturbed. Here, we identified conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to global accumulation of proteins and increased cytoplasmic density. Upon removal of these perturbations, this biomass accumulation drove cells to undergo a multi-generational period of “supergrowth” in which rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a novel mechanism for global proteome homeostasis based on modulation of volume growth and dilution.

Cell growth is a complex process in which the biosynthesis of cellular components must be coordinated with increases in cell size in order to maintain their concentrations. In many cell types, the steady-state growth rate is proportional to cell size such that larger cells grow proportionally faster than smaller cells. Little is known about the mechanisms coordinating biosynthesis, cell volume, and cytoplasmic density. Here, we reveal a global mechanism for proteome homeostasis by transiently decoupling protein synthesis from volume growth rate in the fission yeast Schizosaccharomyces pombe. Rapid osmotic oscillations strongly inhibited growth, but when oscillations were terminated cells subsequently underwent a period of "supergrowth" in which growth was significantly faster than steady-state growth for multiple generations. Protein concentrations and cytoplasmic density increased steadily during oscillations, and gradually decreased to steady-state values during supergrowth. Transient growth inhibition by disabling the secretory pathway produced similar behaviors to osmotic oscillations. The accumulation of biomass was responsible for driving subsequent rapid growth, yielding a homeostatic mechanism for maintaining global protein concentration.

A central question in mechanobiology is how cellular-scale structures are established and regulated. In bacteria, the cell envelope is essential for mechanical integrity, protecting against environmental stresses and bearing the load from high turgor pressures.A central question in mechanobiology is how cellular-scale structures are established and regulated. In bacteria, the cell envelope is essential for mechanical integrity, protecting against environmental stresses and bearing the load from high turgor pressures. Trivedi et al. (mBio 9:e01340-18, 2018, https://doi.org/10.1128/mBio.01340-18) screened a Pseudomonas aeruginosa transposon library and identified genes that influence cell stiffness by measuring cell growth while cells were embedded in an agarose gel. Their findings provide a broad knowledge base for how biochemical pathways regulate cellular mechanical properties in this pathogen. Dozens of genes across diverse functional categories were implicated, suggesting that cellular mechanics is a systems-level emergent property. Furthermore, changes in d-alanine levels in a dadA (d-alanine dehydrogenase) mutant resulted in decreases in the expression of cell wall enzymes, cross-linking density, and cell stiffness. These insights into the biochemical and mechanical roles of dadA highlight the importance of systems-level investigations into the physical properties of cells.

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of tumor overexpressed gene (TOG) domains, yet their mechanism remains unknown. Here, we describe the biochemical and structural basis for TOG arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 subunits, in which each domain exhibits a distinct exchange rate for tubulin. X-ray structures revealed square-shaped assemblies composed of pseudo-dimeric TOG1-TOG2 subunits assembled head-to-tail, positioning four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking and electron microscopy show Alp14-tubulin forms square assemblies in solution, and inactivating their interfaces destabilize this organization without influencing tubulin binding. An X-ray structure determined using approach to modulate tubulin polymerization revealed an unfurled assembly, in which TOG1-TOG2 uniquely bind to two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies that then unfurl, facilitating their concerted polymerization into protofilaments.

2.5D assembly, a package with stacking GPU and HBM chips onto through silicon via interposer (TSI), then mounted onto organic substrate is now mass produced and dedicated package solution for high performance and large volume numerical calculation applications. However, high cost for TSI wafer and substrate, also the large/thick package dimension and long assembly cycle time of 2.5D package are also limit its penetration of high performance applications. The applications using 2.5D package are continuously drive OSATs to thinner the outline dimension and assembly cycle time, especially the cost of 2.5D package, wish to apply this assembly platform on a wider field.A directly derivative of 2.5D package with thinner thickness/cost is to eliminate the organic substrate, and make the function of organic substrate as an RDL circuits onto the TSV interposer, so called TSI-FCBGA (patented). As a package without organic substrate, the outline dimension of this package will be only half of original structure. It will also shorten the cycle time of secondary assembly processes of chip modules onto organic substrate. As to eliminate organic substrate, the original at least 6 layers circuits on it is now re-distributed onto TSV interposer will make the RDL circuits on TSV interposer with fine line space and pitch to ensure matchable I/O before and after eliminating the organic substrate. Thus, the interconnection solder ball size and pitch will be smaller than that of original package with organic substrate. However, there is no reliability data available for this TSI-FCBGA package with smaller solder ball size and pitch.For the work of this paper, we modified platform of 2.5D package chip module with changing C4 side micro bump to solder ball, as a test vehicle of TSI-FCBGA, and demonstrated the board level reliability of it with different solder ball type, size, pitch, also molded solder ball on TSV interposer with daisy chain circuits. The results show the board level reliability of this thinner 2.5D package can pass mostly 733 thermal cycles with definite solder ball types and properties, though it is a little worse than original 2.5D package with organic substrate. Simulations are furtherly executed to find better structure of UBM and RDLs for future study.

The XMAP215/Stu2/Alp14 microtubule polymerases utilize Tumor Overexpressed Gene (TOG) domain arrays to accelerate microtubule plus-end polymerization. Structural studies suggest a microtubule polymerase model in which TOG arrays recruit four αβ-tubulins, forming large square assemblies; an array of TOG1 and TOG2 domains may then unfurl from the square state to polymerize two αβ-tubulins into protofilaments at microtubule ends. Here, we test this model using two biochemically characterized classes of fission yeast Alp14 mutants. Using in vitro reconstitution and in vivo live cell imaging, we show that αβ-tubulins recruited by TOG1 and TOG2 domains serve non-additive roles in microtubule plus-end tracking and polymerase activities. Alp14 mutants with inactivated square assembly interfaces have defects in processive plus-end tracking and poor microtubule polymerase, indicating a functional role for square assemblies in processive tracking. These studies provide functional insights into how TOG1 and TOG2 domain arrays recruit tubulins and promote polymerase at microtubule plus ends.

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of Tumor Overexpressed Gene (TOG) domains. The underlying mechanism of these arrays as microtubule polymerases remains unknown. Here, we describe the biochemical and structural basis for TOG domain arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 arrays, each with distinct exchange rates. X-ray structures reveal pseudo-dimeric square-shaped assemblies in which four TOG domains position four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking confirms square assemblies form in solution, and inactivation of their interfaces destabilizes square organizations without influencing tubulin binding. Using an approach to modulate tubulin polymerization, we determined a X-ray structure showing an unfurled assembly in which TOG1 and TOG2 uniquely bind two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies, which then unfurl facilitating their concerted polymerization into protofilaments.

The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro, it is not yet clear whether these proteins participate in MT nucleation in vivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT., This work was supported by NIH grantsR01GM069670 andR01GM115185 (to F.C.) and by anMEC/Fulbright postdoctoral award (FMECD-2011/6545641100), the Andalucía Talent Hub Program/Marie Skłodowska-Curie actions (COFUND; grant agreement no. 291780), and funding from the research program of Universidad Pablo de Olavide (to I.F.-P.).

A 32Gb/s-NRZ, 15GBaud/s-PAM4 configurable DFB Laser Diode Driver (LDD) is presented in standard 65nm CMOS. The driver employs a balanced-input, single-ended-output topology to deliver large current output, and integrates a tunable pre-emphasis to extend the bandwidth. An on-chip active back-termination (ABT) is proposed which absorbs loading reflections without sacrificing the effective modulation current. Directly wire-bonded to a DFB laser chip, the measurement results show 25.78Gb/s NRZ optical eye-diagram with 4dB Extinction Ratio (ER) and a 19.3% eye-mask margin referred to the 100G specification. The LDD delivers 60mA bias and 60mApp modulation current, consuming only 550mW power consumption.

One of the major challenges of modern cell biology is to understand how cells are assembled from nanoscale components into micrometer-scale entities with a specific size and shape. Here I describe how our quest to understand the morphogenesis of the fission yeast Schizosaccharomyces pombe drove us to investigate cellular mechanics. These studies build on the view that cell shape arises from the physical properties of an elastic cell wall inflated by internal turgor pressure. Consideration of cellular mechanics provides new insights into not only mechanisms responsible for cell-shape determination and growth, but also cellular processes such as cytokinesis and endocytosis. Studies in yeast can help to illuminate approaches and mechanisms to study the mechanobiology of the cell surface in other cell types, including animal cells.

Objectives: Whiplash-associated disorders (WADs), or whiplash injuries, due to low-severity vehicle crashes are of great concern in motorized countries and it is well established that the risk of such injuries is higher for females than for males, even in similar crash conditions. Recent protective systems have been shown to be more beneficial for males than for females. Hence, there is a need for improved tools to address female WAD prevention when developing and evaluating the performance of whiplash protection systems. The objective of this study is to develop and evaluate a finite element model of a 50th percentile female rear impact crash test dummy.Methods: The anthropometry of the 50th percentile female was specified based on literature data. The model, called EvaRID (female rear impact dummy), was based on the same design concept as the existing 50th percentile male rear impact dummy, the BioRID II. A scaling approach was developed and the first version, EvaRID V1.0, was implemented. Its dynamic response was compared to female volunteer data from rear impact sled tests.Results: The EvaRID V1.0 model and the volunteer tests compared well until ∼250 ms of the head and T1 forward accelerations and rearward linear displacements and of the head rearward angular displacement. Markedly less T1 rearward angular displacement was found for the EvaRID model compared to the female volunteers. Similar results were received for the BioRID II model when comparing simulated responses with experimental data under volunteer loading conditions. The results indicate that the biofidelity of the EvaRID V1.0 and BioRID II FE models have limitations, predominantly in the T1 rearward angular displacement, at low velocity changes (7 km/h). The BioRID II model was validated against dummy test results in a loading range close to consumer test conditions (EuroNCAP) and lower severity levels of volunteer testing were not considered.Conclusions: The EvaRID dummy model demonstrated the potential of becoming a valuable tool when evaluating and developing seats and whiplash protection systems. However, updates of the joint stiffness will be required to provide better correlation at lower load levels. Moreover, the seated posture, curvature of the spine, and head position of 50th percentile female occupants needs to be established and implemented in future models.

XMAP215/Dis1 proteins are microtubule polymerases that accelerate αβ-tubulins assembly at microtubule plus ends via multiple conserved Tumor Overexpressed Gene (TOG) domains; however, their mechanisms remains poorly understood. Here, we describe structural basis for microtubule polymerase using biochemistry and x-ray crystallography by studying multi-TOG domain-αβ-tubulin complexes in two states along the polymerization pathway. We show TOG1 and TOG2 domains bind αβ-tubulins but release them with different rates using biochemical methods. Upon inhibiting αβ-tubulin polymerization, we determine x-ray structures revealing that two subunits of TOG1-TOG2 domains form dimeric head-to-tail square assemblies bound to αβ-tubulins, stabilized by novel higher-order TOG domain interfaces. In this intermediate the four αβ-tubulins are positioned in a polarized organization, 90°-rotated from polymerization contacts. Upon controlled release of αβ-tubulin polymerization, TOG2 dissociate from TOG1 domains, rotating and polymerizing αβ-tubulins to form a head-to-tail two-subunit polymer. A new paradigm emerges from our studies to explain microtubule polymerase catalysis suggesting two TOG1-TOG2 domain subunits cooperatively organize αβ-tubulins in a polarized organization prior to polymerization, and dissociation of TOG domains at microtubule plus ends leads tubulin polymerization due to the organization coupled with an αβ-tubulin TOG domain release rate gradient, with the most rapid being at the outermost end of a newly formed polymer.

Csi1 promotes centromere clustering by linking centromeres to the SUN domain protein Sad1 in the nuclear envelope., In the fission yeast Schizosaccharomyces pombe, the centromeres of each chromosome are clustered together and attached to the nuclear envelope near the site of the spindle pole body during interphase. The mechanism and functional importance of this arrangement of chromosomes are poorly understood. In this paper, we identified a novel nuclear protein, Csi1, that localized to the site of centromere attachment and interacted with both the inner nuclear envelope SUN domain protein Sad1 and centromeres. Both Csi1 and Sad1 mutants exhibited centromere clustering defects in a high percentage of cells. Csi1 mutants also displayed a high rate of chromosome loss during mitosis, significant mitotic delays, and sensitivity to perturbations in microtubule–kinetochore interactions and chromosome numbers. These studies thus define a molecular link between the centromere and nuclear envelope that is responsible for centromere clustering.

The mechanism by which spindle microtubules (MTs) determine the site of cell division in animal cells is still highly controversial. Putative cytokinesis “signals” have been proposed to be positioned by spindle MTs at equatorial cortical regions to increase cortical contractility and/or at polar regions to decrease contractility [Rappaport, 1986; von Dassow, 2009]. Given the relative paucity of MTs at the future division site, it has not been clear how MTs localize cytokinesis factors there. Here, we test cytokinesis models using computational and experimental approaches. We present a simple lattice-based model in which signal-kinesin complexes move by transient plus-end directed movements on MTs interspersed with occasions of uniform diffusion in the cytoplasm. In simulations, complexes distribute themselves initially at the spindle midzone and then move on astral MTs to accumulate with time at the equatorial cortex. Simulations accurately predict cleavage patterns of cells with different geometries and MT arrangements and elucidate several experimental observations that have defied easy explanation by previous models. We verify this model with experiments on indented sea urchin zygotes showing that cells often divide perpendicular to the spindle at sites distinct from the indentations. These studies support an equatorial stimulation model and provide a simple mechanism explaining how cytokinesis factors localize to the future division site. © 2012 Wiley Periodicals, Inc

Summary Chemical gradients can generate pattern formation in biological systems. In the fission yeast Schizosaccharomyces pombe , a cortical gradient of pom1p (a DYRK-type protein kinase) functions to position sites of cytokinesis and cell polarity and to control cell length. Here, using quantitative imaging, fluorescence correlation spectroscopy, and mathematical modeling, we study how its gradient distribution is formed. Pom1p gradients exhibit large cell-to-cell variability, as well as dynamic fluctuations in each individual gradient. Our data lead to a two-state model for gradient formation in which pom1p molecules associate with the plasma membrane at cell tips and then diffuse on the membrane while aggregating into and fragmenting from clusters, before disassociating from the membrane. In contrast to a classical one-component gradient, this two-state gradient buffers against cell-to-cell variations in protein concentration. This buffering mechanism, together with time averaging to reduce intrinsic noise, allows the pom1p gradient to specify positional information in a robust manner.

Alp14, a XMAP215 orthologue in fission yeast, is a microtubule (MT) polymerase. It tracks growing MT plus ends and regulates the polymerization state of tubulin by cycling between a tubulin dimer–bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly., XMAP215/Dis1 proteins are conserved tubulin-binding TOG-domain proteins that regulate microtubule (MT) plus-end dynamics. Here we show that Alp14, a XMAP215 orthologue in fission yeast, Schizosaccharomyces pombe, has properties of a MT polymerase. In vivo, Alp14 localizes to growing MT plus ends in a manner independent of Mal3 (EB1). alp14-null mutants display short interphase MTs with twofold slower assembly rate and frequent pauses. Alp14 is a homodimer that binds a single tubulin dimer. In vitro, purified Alp14 molecules track growing MT plus ends and accelerate MT assembly threefold. TOG-domain mutants demonstrate that tubulin binding is critical for function and plus end localization. Overexpression of Alp14 or only its TOG domains causes complete MT loss in vivo, and high Alp14 concentration inhibits MT assembly in vitro. These inhibitory effects may arise from Alp14 sequestration of tubulin and effects on the MT. Our studies suggest that Alp14 regulates the polymerization state of tubulin by cycling between a tubulin dimer–bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly.

SummaryElectric signals surround tissues and cells and have been proposed to participate in directing cell polarity in processes such as development, wound healing, and host invasion [1, 2]. The application of exogenous electric fields (EFs) can direct cell polarization in cell types ranging from bacteria and fungi to neurons and neutrophils [3–7]. The mechanisms by which EFs modulate cell polarity, however, remain poorly understood. Here we introduce the fission yeast Schizosaccharomyces pombe as a model organism to elucidate the mechanisms underlying this process. In these rod-shaped cells, an exogenous EF reorients cell growth in a direction orthogonal to the field, producing cells with a bent morphology. A candidate genetic screen identifies conserved factors involved in this process: an integral membrane proton ATPase pma1p that regulates intracellular pH, the small GTPase cdc42p, and the formin for3p that assembles actin cables. Interestingly, mutants in these genes still respond to the EF but orient in a different direction, toward the anode. In addition, EFs also cause electrophoretic movement of cell wall synthase complex proteins toward the anode. These data suggest molecular models for how the EF reorients cell polarization by modulating intracellular pH and steering cell polarity factors in multiple directions.