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Athale, C. A.

Publications and source records attributed to Athale, C. A..

14 recordsLinked to original sources

Coin-tossing by Kinesin-1 Head and Tail Binding Collectively Drives Microtubule Patterns

Intracellular microtubule-based transport depends on an essential plus-ended molecular motor, kinesin-1. The N-terminal ATP-dependent head driving motility and a C-terminal cargo interacting tail, both bind microtubules. Previously, the interplay of both domains were hypothesized to play a role in collective microtubule sliding patterns, but the mechanisms remained unclear. Here, we show that full-length Drosophila kinesin-1 generates striking spatiotemporal patterns in gliding assays including bending, looping, and oscillations as well as stop-and-go motion of filaments. In presence of the motor domain alone these patterns are absent, but with the tail only construct, microtubules bind passively, with the tail acting as a static anchor. An equimolar mixture of these two domain constructs reproduces these spatio-temporal patterns both in terms of increased bending with length and velocity distributions. Our results support a simple coin-toss model, where either head or tail bind microtubules with equal probability, leading to antagonistic interactions that drives emergent motility patterns and velocity distributions. Thus the kinesin tail plays an active mechanical role beyond regulation or cargo binding, modulating microtubule behavior through inter-domain competition.

biophysics↗

Mechanics of E. coli cell width homeostasis and bulging dynamics from MreB and septum inhibition

The mechanobiology of cytoskeleton and cell envelope play a vital role in cell shape homeostasis. In the gram-negative model rod-shaped bacterium Escherichia coli antibiotics that weaken the cell envelope are seen to result in bulges that eventually lead to lysis. Here, we quantify the shape dynamics of E. coli treated with cephalexin, a PBP3 inhibitor and A22, an MreB polymerization inhibitor from single cell microscopy. We find low concentrations of both inhibitors result in bulge formation with multiple cell shapes observed: rugby, large bacilli and rods with two and three bulges. We quantify the parameters of cell envelope rigidity, pressure and fluidity obtained from fitting a computational model of shell-mechanics to length and width dynamics from experiment. Using this optimized model we estimate the turgor and growth pressures of untreated growing cells as {approx}0.15 MPa and {approx}0.4 MPa. The bulge expansion dynamics correlate most prominently with a change in bending rigidity of the cell wall and cytoskeleton. Simulations predict a threshold behavior in response to envelope bending rigidity that is validated by comparison with experiments of E. coli treated with A22 either in isolation or in combination, resulting in loss of width control and cell shape change.

cell biology↗

Deep learning-based reconstruction of embryonic cell-division cycle from label-free microscopy time-series of evolutionarily diverse nematodes

Microscopy of cellular dynamics during embryogenesis of non-model organisms can be tech- nically challenging due to limitations of molecular labelling methods. Label-free differential interference contrast (DIC) microscopy of the first embryonic cell division of nematodes related to Caenorhabditis elegans has been successfully employed to examine the constraints and divergence of intra-cellular mechanisms during this asymmetric cell division. However, identifying stages of the cell division cycle were performed interactively, pointing to a need to automate of cell stage identification from DIC microscopy. To this end, we have trained deep convolutional neural networks (CNNs), both pre-existing such as ResNet, VGGNet and EfficientNet, and a customized shallow network, EvoCellNet, to automatically classify first-embryonic division into the stages: (i) pro-nuclear migration and (ii) centration and rotation, (iii) spindle elongation and (iv) cytokinesis, with all networks performing with 91% or greater accuracy. The activations of the networks superimposed on the images result in segmentation-free detection of intracellular features such as pro-nuclei, spindle and spindle- poles in case of the shallow EvoCellNet, while ResNet, VGGNet and and EfficientNet detect large-scale, features that are less biologically meaningful. The UMAP space representation combined with support vector machines (SVM) allows for stage boundary identification and recovers a cyclical map connecting the states (i) to (iv) of the division. This approach could be used to automate quantification of cell division stages and sub-cellular dynamics without explicit labelling in label-free microscopy. SummaryWe have trained multiple convolutional neural networks (CNNs) to classify the stages of cell division from the first embryonic division of diverse nematodes, evolutionarily related to Caenorhabditis elegans. We find two classifiers, VggNet and a customized EvoCellNet, can detect intracellular features and a UMAP representation can reconstruct the cyclical progression of first embryonic division from related species.

developmental biology↗

Microtubule polymerization tuned by macromolecular crowdant size and density

Microtubule (MT) polymerization is regulated by biochemical as well as physical factors such as macromolecular crowding. Crowding agents or crowdants affect MT elongation rates differently depending on crowdant size due to opposing ef-fects on polymerization: microviscosity reduces polymer elongation, while volume exclusion increases reaction rates by local concentration. In order to address how crowdant size and concentration collectively affect MT populations, we combine in vitro MT polymerization experiments with kinetic Monte Carlo simulations. Our experiments in bulk with nucleators validate decreasing MT elongation rates with increasing concentrations of small molecular weight crowdants in bulk assays and a corresponding increase for large crowdants. Kinetic Monte Carlo simulations can explain the result with packing fractions dependence of small as compared to large crowdants increasing microviscosity more dramatically. In contrast MT bulk polymerization rates in absence of nucleators increased with crowdant con-centration, irrespective of their size, with a corresponding decrease in the critical concentration. Microscopy of filament growth dynamics demonstrates that small crowdants result in shorter filaments in a concentration dependent manner, consis-tent with their role in reducing elongation rates, but this decrease is compensated by increased number of filaments. Large crowdants increase the filament numbers while elongation is slightly decreased. Our results provide evidence for MT nucle-ation being rate-limited and elongation diffusion limited, resulting in differences in the effect of crowdant sizes on nucleation and elongation. These results are of gen-eral relevance to understand physical effects of crowding on collective cytoskeletal polymerization dynamics.

biophysics↗

Multi-protein chimeric antigens, a novel combined approach for efficiently targeting and blocking the blood stage of Plasmodium falciparum

Plasmodium falciparum-induced malaria remains a fatal disease affecting millions of people worldwide. Mainly, the blood stage of malaria is highly pathogenic and symptomatic, rapidly damaging the host organs and occasionally leading to death. Currently, no vaccines are approved for use against the blood stage of malaria. Canonical vaccines in the past have selected the most immunodominant or essential protein to block the growth of the parasite. This strategy works efficiently for low-complexity organisms such as viruses and a few bacteria but has not shown promising results for a malaria vaccine. Plasmodium has a complex life cycle and vaccine candidates especially during blood stage are ineffective due to multiple gene families showing redundancy, immune evasion, and insufficient antibody titer. Herein, we demonstrate a novel strategy of combining multiple antigens from the blood stage of Plasmodium falciparum using only the most immunodominant peptide sequences as a way of tackling polymorphism and redundancy. We created three chimeric antigens targeting eight PfEMP1 proteins (chimeric varB) and eight merozoite surface proteins (chimeric MSP and InvP) by selecting and stitching B-cell epitopes. Our chimeric constructs show naturally circulating antibodies against individual peptides using epitope-mapping microarray as well as entire proteins in malaria-infected patients. We demonstrate that anti-varB antibodies are neutralizing in nature and significantly reduce the cytoadhesion on an organ-on-chip system with a microfluidic device mimicking physiological conditions. We have applied a Deep Learning based method to quantify the number of adhered RBCs under fluidic conditions that is used to study cytoadhesion. Furthermore, the anti-MSP and InvP antibodies show complete growth inhibition in a single cycle at a combined concentration of 0.13 mg/ml. Overall, our results show that a combination of antigenic peptides from multiple antigens can function as a next-generation vaccine and effectively block the blood stage by reducing cytoadhesion and inhibiting the parasite growth.

molecular biology↗

Effect of Size Variability, Filamentation and Division Dynamics on Escherichia coli Allometry

The cell surface area (SA) increase with volume (V) for cells is determined by growth and regulation of size and shape. Most studies of the rod-shaped model bacterium Escherichia coli have focussed on the phenomenology or molecular mechanisms governing such scaling. Here, we proceed to examine the role of population statistics and cell division dynamics in such scaling by a combination of microscopy, image analysis and statistical simulations. We find that while cells sampled from mid-log cultures follow a 2/3 exponent, similar to geometric (Platonic) solids, drug induced filamentous cells have higher exponent values. Modulating the growth rate to change the proportion of filamentous cells, we find SA-V scales with an exponent > 2/3, exceeding that predicted by the geometric scaling law. However, since increasing growth rates alter the mean and spread of population cell size distributions, we use statistical modeling to disambiguate between the effect of the mean size and variability. Simulating (i) increasing mean cell length with a constant standard deviation (s.d.) and (ii) a constant mean length with increasing s.d. results in scaling exponents that exceed the 2/3 geometric law, when population variability is included. In order to overcome possible effects of statistical sampling of unsynchronized cell populations, we virtually synchronized the estimation of SA-V scaling from single cell growth experiments. We find the exponent depends on the stage with the maximal cell length heterogeneity and scaling exponent observed during the intermediate period between birth (B) and division (D) stages. These results point to a need to consider population statistics and a role for cell growth and division when estimating SA-V scaling of bacterial cells.

biophysics↗

Emergence of flagella-like oscillations in single microtubules driven by collective dynein transport

Flagellar and ciliary oscillations result from a combination of stereotypical axonemal geometry, collective mechanics of motors, microtubules (MTs), elastic linkers and biochemical regulation. However, the minimal essential components and constraints resulting in flagellar oscillations remain unclear. Here, we demonstrate that periodic, low-frequency waves of flagella-like oscillations in vitro emerge from a ATP-driven collective molecular motor transport of MTs clamped at one end. The spontaneous oscillations arise without any external forcing and can be explained by an in silico model of molecular motor binding driven MT bending and buckling followed by motor detachment driven recovery stroke. We demonstrate that transitions in single MT patterns between flapping, flagellar-beating and looping are determined solely by the self-organization of collective motor transport and filament elasticity.

biophysics↗

Cell size and shape regulation of E. coli determines surface area scaling with volume

The scaling of surface area and volume of cells has widespread consequences for cell physiology, growth and adaptation. While the surface area increases with volume as SA ~ V{gamma} the scaling exponent for proportional growth maintaining the shape and aspect is {gamma} ~ 2/3 or 0.66. However most well-studied cellular systems deviate from this standard exponent. At the same time, a mechanism that could predict the biological or physical basis of these scaling relations remains unclear. Here, we quantify the surface area scaling with volume of Escherichia coli cells with varying growth rates and under different conditions and find the scaling exponent varies from {gamma} ~ 0.7 to 0.9. A model of uncorrelated statistical variation of cell lengths and widths can reproduce the exponent observed in experiment. Average values of length and width on the other hand results in an impression of ideal geometric scaling, as reported in some studies. Experimental data however suggests that E. coli cell width is strongly correlated with length and a model of saturation best explains the observations. We hypothesize this model of cell size and shape regulation could serve the function of optimizing flux of nutrients, within the constraints of the cell division machinery.

microbiology↗

Evolutionary divergence of anaphase spindle mechanics in nematode embryos constrained by antagnonistic pulling and viscous forces

Cells are the basic unit of biological organization, and their division is remarkably conserved across phyla. However from an evolutionary perspective, it remains unclear how much cellular parameters can diverge, without altering the basic function they sustain. We address the mechanics of asymmetric mitotic spindle positioning during the first embryonic division of six nematode species. We propose a viscoelastic model of spindle positioning and mobility that can provide a physical explanation of why in C. elegans it undergoes oscillations during elongation, whereas most others lack oscillations. To test this model, we measured the pulling forces and opposing cytoplasmic drag by a combination of laser ablation of the anaphase spindle and tracking of intracellular granules. While centrosomes of all species recoil on spindle cutting, quantitative differences correlate with the cytoplasmic viscosity. In fact, increased viscosity correlates with decreased oscillation speeds of intact spindles across species. However, the absence of oscillations despite low viscosity in some species, can only be explained by smaller pulling forces. Consequently, we find that spindle mobility across the species analyzed here is characterized by a tradeoff between cytoplasmic viscosity and pulling forces. Our work provides a framework for understanding mechanical constraints on evolutionary diversification of spindle mobility.

biophysics↗

Effect of plant tubulin kinetic diversification on microtubule lengths

Microtubules (MTs) are dynamic polymers vital for cellular physiology. Bulk tubulin polymerization is nucleation dependent, while individual filaments exhibit dynamic instability driven by GTP hydrolysis rates. Although MTs assembled from well-studied animal brain tubulins have very comparable nucleation and GTP-hydrolysis rates, the kinetic rates of evolutionarily more distant species could diverge. Here we focus on a plant tubulin, the legume Vigna sp. (mung bean) to test the effect of kinetic diversification on MT polymerization. We activity purify tubulin from seedlings and find MT filaments are fewer and shorter than animal brain tubulin. We find mung tubulin polymerization kinetics is nucleation dependent with a high rate of GTP hydrolysis and a critical concentration lower than previously reported for tubulins. A computational model of the kinetics based on the relative influence of rates of nucleation and hydrolysis demonstrates increased rates of hydrolysis can affect MT filament numbers and their lengths, as compared to increasing nucleation rates. Our approach provides a framework to compare the effect of evolutionary diversification of MT nucleation and elongation.

biophysics↗

Dynein collective behavior in mitotic nuclear positioning of Saccharomyces cerevisiae

1Positioning the nucleus at the bud-neck prior during Saccharomyces cerevisiae mitosis during anaphase involves pulling forces of cytoplasmic dynein localized in the daughter cell. While genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and dyneins numbers driving the process has remained difficult. In order to better understand the role of motor-microtubule mechanics during nuclear positioning and the role of dynein, we have used a computational model of nuclear mobility in S. cerevisiae and reconciled it to the mobility of labelled spindle pole bodies (SPBs) measured by quantifying fluorescence microscopy time-series. We model the apparent random-walk mobility of SPBs by combining diffusion of the nucleus and active pushing of MTs at the cell membrane. By minimizing the deviation between tracks of fluorescently tagged SPBs and simulations, we estimate the effective cytoplasmic viscosity to be 0.5 Pa s. The directed transport of nuclei during the budding process is similarly quantified by tracking the daughter SPB (SPB-D) in experiment. Using force-balance, we find 2 to 8 motors are required to pull the nucleus to the bud-neck. Simulations of the cytoplasmic MT (cMT) search and capture by dynein suggest single motor binding is followed by a rapid saturation of number of bound motors. The short time and length of MT interactions with the cortex and minimal collective dynein force required, predict a functional role for dynein clustering in nuclear positioning.

cell biology↗

Asymmetric Cell Growth and Nucleoid Displacement in Escherichia coli

Single celled growth of Escherichia coli is typically considered as an example symmetric division, based on the sizes of daughter cells and precision of center finding of the septum. Here, we investigate the symmetry of membrane addition in the mid-plane and DNA segregation using a video-microscopy approach. We find the membrane expansion dynamics to be asymmetric based on mid-cell photobleaching landmarks in FM4-64, used to stain the membrane. The apparent growth bias of E. coli does not correspond to the age of the pole. We find the membrane growth asymmetry is correlated to nucleoid displacement, consistent with ideas of coupling of cell growth and nucleoid positioning. The mobility of the actin-homolog MreB also correlates with membrane growth asymmetry, based on fluorescence recovery after photobleaching (FRAP) measurements of a YFP-fusion. These correlations suggest the small asymmetry of membrane addition observed could potentially drive nucleoid segregation and MreB mobility asymmetry in E. coli.\n\nIMPORTANCEAsymmetry in bacterial cell division is seen in its most simple form in the binomial event of protein segregation that results in noise in equal segregation and depends on the protein copy number. In the case of specific proteins this can also affect growth, and result in differentiation. However, during the asexual division of Escherichia coli single cells are thought to grow symmetrically and divide equally. We find a slight but consistent asymmetry in growth based on quantitative morphometry of cell pole displacement in time-series of growing E. coli cells. Increased cell wall extension in one half of the cell over the other appears to explain the asymmetry in the displacement of cell poles. Interestingly the growth asymmetry is not correlated to the age of the pole (old and new). This observed asymmetry appears to correlate with the asymmetry of nucleoid segregation, resulting in the nucleoids finding the midpoints of the respective daughter cells, and formation of the septum at the geometric centre of the elongated cell just prior to division. The mobility dynamics of the cytoskeletal protein MreB, which organizes the cell membrane, is are more dynamic where membrane growth is faster. Thus we propose a linkage between this observed growth asymmetry and that of MreB dynamics.

microbiology↗

‘Switch-like’ transition from random to directed motility of microtubules by a yeast dynein

Processive transport by multiple molecular motors that step stochastically, requires a form of mechanical coupling. In a quantitative microtubule (MT) gliding assay with yeast cytoplasmic dynein, we investigate the nature of this coupling by examining the effect of MT length and motor density on transport. We find speed and velocity have a length dependence for low motor numbers, but are independent of MT length for high motor densities. The dependence of speed, velocity and degree of randomness of MT transport is best understood when evaluated in terms of the numbers of motors bound to a filament. A model of collective transport of MTs, based on stochastic stepping and asymmetric detachment rates, reproduces the experimental trends of decreasing diffusivity with increasing number of motors. Additionally, the model predicts a switch-like increase in directionality of MT transport above a threshold number of motors. Such a rapid transition from random to directed motility with increasing numbers of yeast dyneins, could play a role in vivo during mitosis in the search and orientation of the Saccharomyces cerevisiae nucleus.

biophysics↗

Mechanics of Hydra Detachment from Subrates: The Role of Substrate Rigidity and Starvation

Hydra is a fresh water hydrozoan living as a solitary polyp with a sedentary feeder lifestyle attached to a substrate. In times of food shortage they are reported to detach from their substrate and move either by drifting or somer-saulting. The attachment to the substrate is usually by the basal-body which secretes a mucosal adhesive. The mechanical strength of the adhesion of Hydra has not been quantified so far. Here, we measure the force required to detach Hydra vulgaris and Hydra magnipapillata from a surface and the role of physical and physiological factors. In order to do this, we have developed a flow chamber with a calibrated jet of water. We find H. vulgaris adhering to a hard substrate - a glass cover slip- requires more force to detach it as compared to a soft substrate- polyacrylamide gel. While H. vulgaris after one week of starvation detaches with very similar values of stress, H. magnipapillata detaches more readily when starved. These results suggest that the strength of adhesion is strongly affected by the stiffness of the substrate, while nutritional status dependence of detachment force appears to be species dependent. Given that Hydra detachment is required during locomotion, our measurements on the one hand suggest the magnitude of forces the animal must exert to detach itself. Additionally, our results suggest active detachment of the base might be required for Hydra to achieve movement, and only a small contribution coming from weakening adhesion.

biophysics↗