Mechanics of cell width homeostasis and bulge dynamics from MreB and septum inhibition.

cell shape and size are governed by the mechanochemistry of the cellular components.Inhibiting either cell wall synthesis proteins such as FtsI leads to cell elongation and bulging, while inhibiting MreB cytoskeletal polymerization results in a loss of rod-shape. Here, we quantify cell shape dynamics of combinatorially treated with the FtsI inhibitor cephalexin and MreB inhibitor A22 and fit a shell mechanics model to the length-width dynamics to infer the range of effective mechanical properties governing cell shape.

Classification of first embryonic division stages of multiple Caenorhabditis species by deep learning.

The first embryonic division of Caenorhabditis elegans is a model for asymmetric cell division, and identifying the stages of cell division across related species could improve our understanding of the divergence of cellular events and mechanisms. Comparative microscopy of evolutionarily divergent species continues to rely on label-free differential interference contrast (DIC) microscopy due to technical challenges in molecular tagging, with the identification of cell division stages still relying on label-free microscopy. Here, we compare multiple deep convolutional neural networks (CNNs) trained to automate cell stage classification in DIC microscopy movies and interpret the results, with code and classification weights released as OpenSource.

Translational Enhancer Based Amplification of Toehold Sensors (TacToe) for Improved Sensitivity and Speed of Viral RNA Detection.

Toehold switches are RNA riboswitches activated by complementary nucleic acid sequences that have shown promise as point-of-care (PoC) molecular diagnostics. However, typical implementations require an additional nucleic-acid-sequence-specific amplification step. Here, we describe a novel toehold switch with a T7-g10 translational enhancer (Tac) that amplifies the expression of the reporter gene regulated by toehold (Toe) sensors.

Physical effects of crowdant size and concentration on collective microtubule polymerization.

The polymerization of cytoskeletal filaments is regulated by both biochemical pathways, as well as physical factors such as crowding. The effect of crowding in vivo emerges from the density of intracellular components. Due to the complexity of the intracellular environment, most studies are based on either in vitro reconstitution or theory.

KnotResolver: tracking self-intersecting filaments in microscopy using directed graphs.

Motivation: Quantification of microscopy time series of in vitro reconstituted motor-driven microtubule transport in "gliding assays" is typically performed using computational object tracking tools. However, these are limited to non-intersecting and rod-like filaments.

Results: Here, we describe a novel computational image-analysis pipeline, KnotResolver, to track image time series of highly curved self-intersecting looped filaments (knots) by resolving cross-overs.

Collective effect of Vigna sp. (mung) tubulin GTP hydrolysis rate divergence on microtubule filament assembly.

Microtubules (MTs) are dynamic cytoskeletal filaments with highly conserved sequences across evolution, polymerizing by the GTP-dependent assembly of tubulin subunits. Despite the sequence conservation, MT polymerization kinetics diverge quantitatively between vertebrate brain, the model plant Arabidopsis and the protozoan Plasmodium. Previously, tubulin purified from seedlings of the plant Vigna sp.

Quantification of cell shape, intracellular flows and transport based on DIC object detection and tracking.

Computational image analysis combined with label-free imaging has helped maintain its relevance for cell biology, despite the rapid technical improvements in fluorescence microscopy with the molecular specificity of tags. Here, we discuss some computational tools developed in our lab and their application to quantify cell shape, intracellular organelle movement and bead transport in vitro, using differential interference contrast (DIC) microscopy data as inputs. The focus of these methods is image filtering to enhance image gradients, and combining them with segmentation and single particle tracking (SPT).

Wave-like oscillations of clamped microtubules driven by collective dynein transport.

Microtubules (MTs) are observed to move and buckle driven by ATP-dependent molecular motors in both mitotic and interphasic eukaryotic cells as well as in specialized structures such as flagella and cilia with a stereotypical geometry. In previous work, clamped MTs driven by a few kinesin motors were seen to buckle and occasionally flap in what was referred to as flagella-like motion. Theoretical models of active-filament dynamics and a following force have predicted that, with sufficient force and binding-unbinding, such clamped filaments should spontaneously undergo periodic buckling oscillations.

Allometry ofsurface area with volume: effect of size variability, filamentation and division dynamics.

The cell surface area (SA) increase with volume (V) is determined by growth and regulation of size and shape. Most studies of the rod-shaped model bacteriumhave 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.

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

Cellular functions such as cell division are remarkably conserved across phyla. However, the evolutionary principles of cellular organization that drive them are less well explored. Thus, an essential question remains: to what extent do cellular parameters evolve without altering the basic functions they sustain? Here we have observed six different nematode species for which the mitotic spindle is positioned asymmetrically during the first embryonic division.

Polymerization kinetics of tubulin from mung seedlings modeled as a competition between nucleation and GTP-hydrolysis rates.

Microtubules (MTs) form physiologically important cytoskeletal structures that are assembled by tubulin polymerization in nucleation- and guanosine triphosphate (GTP)-dependent manner. GTP hydrolysis competes with the addition of monomers, to determine the GTP-cap size, and the onset of shrinkage, which alternates with growth. Multiple theoretical models of MT polymerization dynamics have been reconciled to the kinetics of animal brain tubulins, but more recently, rapid kinetics seen in Arabidopsis tubulin polymerization suggest the need to sample a wider diversity in tubulin polymerization kinetics and reconcile it to theory.

Self-organized optimal packing of kinesin-5-driven microtubule asters scales with cell size.

Radial microtubule (MT) arrays or asters determine cell geometry in animal cells. Multiple asters interacting with motors, such as those in syncytia, form intracellular patterns, but the mechanical principles behind this are not clear. Here, we report that oocytes of the marine ascidian Phallusia mammillata treated with the drug BI-D1870 spontaneously form cytoplasmic MT asters, or cytasters.

Collective dynein transport of the nucleus by pulling on astral microtubules during Saccharomyces cerevisiae mitosis.

Positioning the nucleus at the bud neck during Saccharomyces cerevisiae mitosis involves pulling forces of cytoplasmic dynein localized in the daughter cell. Although genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and the number of dyneins driving the process has remained difficult. To better understand the collective forces involved in nuclear positioning, we compare a model of dyneins-driven microtubule (MT) pulling, MT pushing, and cytoplasmic drag to experiments.

Quantifying Intracellular Particle Flows by DIC Object Tracking.

Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native, unperturbed states with minimal exposure to light. The development of robust computational image-analysis routines is vital to quantitative label-free imaging. The reliability of quantitative analysis of time-series microscopy data based on single-particle tracking relies on accurately detecting objects as distinct from the background, i.

Aster swarming by symmetry breaking of cortical dynein transport and coupling kinesins.

Microtubule (MT) radial arrays or asters establish the internal topology of a cell by interacting with organelles and molecular motors. We proceed to understand the general pattern forming potential of aster-motor systems using a computational model of multiple MT asters interacting with motors in cellular confinement. In this model dynein motors are attached to the cell cortex and plus-ended motors resembling kinesin-5 diffuse in the cell interior.

Collective effects of yeast cytoplasmic dynein based microtubule transport.

Teams of cortically anchored dyneins pulling at microtubules (MTs) are known to be essential for aster, spindle and nuclear positioning during cell division and fertilization. While the single-molecule basis of dynein processivity is now better understood, the effect of increasing numbers of motors on transport is not clear. Here, we examine the collective transport properties of a Saccharomyces cerevisiae cytoplasmic dynein fragment, widely used as a minimal model, by a combination of quantitative MT gliding assays and stochastic simulations.

Role of spatial inhomogenity in GPCR dimerisation predicted by receptor association-diffusion models.

G protein-coupled receptor (GPCR) association is an emerging paradigm with far reaching implications in the regulation of signalling pathways and therapeutic interventions. Recent super resolution microscopy studies have revealed that receptor dimer steady state exhibits sub-second dynamics. In particular the GPCRs, muscarinic acetylcholine receptor M (M1MR) and formyl peptide receptor (FPR), have been demonstrated to exhibit a fast association/dissociation kinetics, independent of ligand binding.

Threshold effect of growth rate on population variability of cell lengths.

A long-standing question in biology is the effect of growth on cell size. Here, we estimate the effect of growth rate () on population cell size distributions by estimating the coefficient of variation of cell lengths (CV) from image analysis of fixed cells in DIC microscopy. We find that the CV is constant at growth rates less than one division per hour, whereas above this threshold, CV increases with an increase in the growth rate.

Automated Multi-Peak Tracking Kymography (AMTraK): A Tool to Quantify Sub-Cellular Dynamics with Sub-Pixel Accuracy.

Kymographs or space-time plots are widely used in cell biology to reduce the dimensions of a time-series in microscopy for both qualitative and quantitative insight into spatio-temporal dynamics. While multiple tools for image kymography have been described before, quantification remains largely manual. Here, we describe a novel software tool for automated multi-peak tracking kymography (AMTraK), which uses peak information and distance minimization to track and automatically quantify kymographs, integrated in a GUI.

A Motor-Gradient and Clustering Model of the Centripetal Motility of MTOCs in Meiosis I of Mouse Oocytes.

Asters nucleated by Microtubule (MT) organizing centers (MTOCs) converge on chromosomes during spindle assembly in mouse oocytes undergoing meiosis I. Time-lapse imaging suggests that this centripetal motion is driven by a biased 'search-and-capture' mechanism. Here, we develop a model of a random walk in a drift field to test the nature of the bias and the spatio-temporal dynamics of the search process.

What can simulations tell us about GPCRs: Integrating the scales.

The functional dynamics of G protein-coupled receptors (GPCRs) encompasses multiple spatiotemporal scales, ranging from femtoseconds to seconds and Ångströms to micrometers. Computational approaches, often in close collaboration with experimental methods, have been invaluable in unraveling GPCR structure and dynamics at these various hierarchical levels. The binding of natural and synthetic ligands to the wild-type and naturally occurring variant receptors have been analyzed by several computational methods.

Thermodynamic and kinetic characterization of transmembrane helix association.

The transient dimerization of transmembrane proteins is an important event in several cellular processes and computational methods are being increasingly used to quantify their underlying energetics. Here, we probe the thermodynamics and kinetics of a simple transmembrane dimer to understand membrane protein association. A multi-step framework has been developed in which the dimerization profiles are calculated from coarse-grain molecular dynamics simulations, followed by meso-scale simulations using parameters calculated from the coarse-grain model.

Collective behavior of minus-ended motors in mitotic microtubule asters gliding toward DNA.

Microtubules (MTs) nucleated by centrosomes form star-shaped structures referred to as asters. Aster motility and dynamics is vital for genome stability, cell division, polarization and differentiation. Asters move either toward the cell center or away from it.

Spatial and temporal sensing limits of microtubule polarization in neuronal growth cones by intracellular gradients and forces.

Neuronal growth cones are the most sensitive among eukaryotic cells in responding to directional chemical cues. Although a dynamic microtubule cytoskeleton has been shown to be essential for growth-cone turning, the precise nature of coupling of the spatial cue with microtubule polarization is less understood. Here we present a computational model of microtubule polarization in a turning neuronal growth cone.