Andrew B Goryachev | University of Edinburgh (original) (raw)

Papers by Andrew B Goryachev

Phys. Rev. E, 2024

We explore a biomimetic model that simulates a cell, with the internal cytoplasm represented by a... more We explore a biomimetic model that simulates a cell, with the internal cytoplasm represented by a
two-dimensional circular domain and the external cortex by a surrounding ring, both modeled using FitzHugh-
Nagumo systems. The external ring is dynamically influenced by a pacemaker-driven wave originating from
the internal domain, leading to the emergence of three distinct dynamical states based on the varying strengths
of coupling. The range of dynamics observed includes phase patterning, the propagation of phase waves, and
interactions between traveling and phase waves. A simplified linear model effectively explains the mechanisms
behind the variety of phase patterns observed, providing insights into the complex interplay between a cell’s
internal and external environments.

Current Biology, 2024

A new analysis of cytokinetic furrow ingression in the Caenorhabditis elegans zygote at high spat... more A new analysis of cytokinetic furrow ingression in the Caenorhabditis elegans zygote at high spatiotemporal
resolution demonstrates that, rather than being a process of steady, spatially uniform constriction, furrow
ingression is modulated by complex contractile oscillations that move around the furrow, possibly in the
form of propagating waves.

Nature Reviews Molecular Cell Biology, 2024

The Rho GTPases-RHOA, RAC1 and CDC42-are small GTP binding proteins that regulate basic biologica... more The Rho GTPases-RHOA, RAC1 and CDC42-are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function.

Proc. Natl. Acad. Sci. USA, 2023

To initiate directed movement, cells must become polarized, establishing a protrusive leading edg... more To initiate directed movement, cells must become polarized, establishing a protrusive leading edge and a contractile trailing edge. This symmetry-breaking process involves reorganization of cytoskeleton and asymmetric distribution of regulatory molecules. However, what triggers and maintains this asymmetry during cell migration remains largely elusive. Here, we established a micropatterning-based 1D motility assay to investigate the molecular basis of symmetry breaking required for directed cell migration. We show that microtubule (MT) detyrosination drives cell polarization by directing kinesin-1-based transport of the adenomatous polyposis coli (APC) protein to cortical sites. This is essential for the formation of cell's leading edge during 1D and 3D cell migration. These data, combined with biophysical modeling, unveil a key role for MT detyrosination in the generation of a positive feedback loop linking MT dynamics and kinesin-1-based transport. Thus, symmetry breaking during cell polarization relies on a feedback loop driven by MT detyrosination that supports directed cell migration.

Journal of Cell Biology, 2022

Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskelet... more Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics.

Molecular Biology of the Cell, 2022

Interest in cortical excitability-the ability of the cell cortex to generate traveling waves of p... more Interest in cortical excitability-the ability of the cell cortex to generate traveling waves of protein activity-has grown considerably over the past 20 years. Attributing biological functions to cortical excitability requires an understanding of the natural behavior of excitable waves and the ability to accurately quantify wave properties. Here we have investigated and quantified the onset of cortical excitability in Xenopus laevis eggs and embryos and the changes in cortical excitability throughout early development. We found that cortical excitability begins to manifest shortly after egg activation. Further, we identified a close relationship between wave properties-such as wave frequency and amplitude-and cell cycle progression as well as cell size. Finally, we identified quantitative differences between cortical excitability in the cleavage furrow relative to nonfurrow cortical excitability and showed that these wave regimes are mutually exclusive.

Journal of Cell Biology, 2022

Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier func... more Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed "Rho flares," but how Rho flares are regulated is unknown. Here, we discovered that intracellular calcium flashes and junction elongation are early events in the Rho flare pathway. Both laser-induced and naturally occurring TJ breaks lead to local calcium flashes at the site of leaks. Additionally, junction elongation induced by optogenetics increases Rho flare frequency, suggesting that Rho flares are mechanically triggered. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSCs) reduces the amplitude of calcium flashes and diminishes the sustained activation of Rho flares. MSC-dependent calcium influx is necessary to maintain global barrier function by regulating reinforcement of local TJ proteins via junction contraction. In all, we uncovered a novel role for MSC-dependent calcium flashes in TJ remodeling, allowing epithelial cells to repair local leaks induced by mechanical stimuli.

Current Biology, 2021

The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic ... more The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division.1,2 During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed “cortical excitability.”3, 4, 5, 6, 7 In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho.7,8 These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation.9 In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics.10 This reconstituted system spontaneously develops two distinct types of self-organized cortical dynamics: singular excitable Rho and F-actin waves, and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the excitable dynamics previously characterized in vivo.7 These findings directly support the long-standing speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics.

Molecular Biology of the Cell, 2021

Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microrid... more Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally elongated protrusions on mucosal epithelial cells, where they form evenly spaced, mazelike patterns that dynamically remodel by fission and fusion. To characterize how microridges form their highly ordered, subcellular patterns and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridge spacing and alignment became increasingly well ordered. Imaging F-actin and non-muscle myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked fission and fusion rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging allowed us to image individual NMII minifilaments in the apical cortex of cells in live animals, revealing that minifilaments are tethered to protrusions and often connect adjacent microridges. NMII minifilaments connecting the ends of two microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. These findings demonstrate that as cells mature, cortical NMII activity orchestrates a remodeling process that creates an increasingly orderly microridge arrangement.

Current Biology, 2021

As the interface between the cell and its environment, the cell cortex must be able to respond to... more As the interface between the cell and its environment, the cell cortex must be able to respond to a variety of external stimuli. This is made possible in part by cortical excitability, a behavior driven by coupled positive and negative feedback loops that generate propagating waves of actin assembly in the cell cortex. Cortical excitability is best known for promoting cell protrusion and allowing the interpretation of and response to chemoattractant gradients in migrating cells. It has recently become apparent, however, that cortical excitability is involved in the response of the cortex to internal signals from the cell-cycle regulatory machinery and the spindle during cell division. Two overlapping functions have been ascribed to cortical excitability in cell division: control of cell division plane placement, and amplification of the activity of the small GTPase Rho at the equatorial cortex during cytokinesis. Here, we propose that cortical excitability explains several important yet poorly understood features of signaling during cell division. We also consider the potential advantages that arise from the use of cortical excitability as a signaling mechanism to regulate cortical dynamics in cell division.

Journal of Cell Biology, 2021

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin fi... more The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.

Soft Matter, 2020

We study the dynamics of pattern formation in a minimal model for active mixtures made of microtu... more We study the dynamics of pattern formation in a minimal model for active mixtures made of microtubules and molecular motors. We monitor the evolution of the (conserved) microtubule density and of the (non-conserved) nematic order parameter, focusing on the effects of an ''anchoring'' term that provides a direct coupling between the preferred microtubule direction and their density gradient. The key control parameter is the ratio between activity and elasticity. When elasticity dominates, the interplay between activity and anchoring leads to formation of banded structures that can undergo additional bending, rotational or splaying instabilities. When activity dominates, the nature of anchoring instead gives rise to a range of active cellular solids, including aster-like networks, disordered foams and spindle-like patterns. We speculate that the introduced ''active model C'' with anchoring is a minimal model to describe pattern formation in a biomimetic analogue of the microtubule cytoskeleton.

Cells, 2020

Cellular morphogenesis is governed by the prepattern based on the symmetry-breaking emergence of ... more Cellular morphogenesis is governed by the prepattern based on the symmetry-breaking emergence of dense protein clusters. Thus, a cluster of active GTPase Cdc42 marks the site of nascent bud in the baker's yeast. An important biological question is which mechanisms control the number of pattern maxima (spots) and, thus, the number of nascent cellular structures. Distinct flavors of theoretical models seem to suggest different predictions. While the classical Turing scenario leads to an array of stably coexisting multiple structures, mass-conserved models predict formation of a single spot that emerges via the greedy competition between the pattern maxima for the common molecular resources. Both the outcome and the kinetics of this competition are of significant biological importance but remained poorly explored. Recent theoretical analyses largely addressed these questions, but their results have not yet been fully appreciated by the broad biological community. Keeping mathematical apparatus and jargon to the minimum, we review the main conclusions of these analyses with their biological implications in mind. Focusing on the specific example of pattern formation by small GTPases, we speculate on the features of the patterning mechanisms that bypass competition and favor formation of multiple coexisting structures and contrast them with those of the mechanisms that harness competition to form unique cellular structures.

Journal of Cell Biology, 2020

Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regul... more Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regulating their formation and arrangement are largely unknown. To study how protrusions form, we focused on the morphogenesis of microridges, elongated actin-based structures that are arranged in maze-like patterns on the apical surfaces of zebrafish skin cells. Microridges form by accreting simple finger-like precursors. Live imaging demonstrated that microridge morphogenesis is linked to apical constriction. A nonmuscle myosin II (NMII) reporter revealed pulsatile contractions of the actomyosin cortex, and inhibiting NMII blocked apical constriction and microridge formation. A biomechanical model suggested that contraction reduces surface tension to permit the fusion of precursors into microridges. Indeed, reducing surface tension with hyperosmolar media promoted microridge formation. In anisotropically stretched cells, microridges formed by precursor fusion along the stretch axis, which computational modeling explained as a consequence of stretch-induced cortical flow. Collectively, our results demonstrate how contraction within the 2D plane of the cortex can pattern 3D cell surfaces.

F1000 Research, 2019

Small GTPases are organizers of a plethora of cellular processes. The time and place of their act... more Small GTPases are organizers of a plethora of cellular processes. The time and place of their activation are tightly controlled by the localization and activation of their regulators, guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Remarkably, in some systems, the upstream regulators of GTPases are also found downstream of their activity. Resulting feedback loops can generate complex spatiotemporal dynamics of GTPases with important functional consequences. Here we discuss the concept of positive autoregulation of small GTPases by the GEF-effector feedback modules and survey recent developments in this exciting area of cell biology.

Soft Matter, 2019

We study the dynamics and phase behaviour of a dry suspension of microtubules and molecular motor... more We study the dynamics and phase behaviour of a dry suspension of microtubules and molecular motors. We obtain a set of continuum equations by rigorously coarse graining a microscopic model where motor-induced interactions lead to parallel or antiparallel ordering. Through numerical simulations, we show that this model generically creates either stable stripes, or a never-settling pattern where stripes periodically form, rotate and then split up. We derive a minimal model which displays the same instability as the full model, and clarifies the underlying physical mechanism. The necessary ingredients are an extensile flux arising from microtubule sliding and an interfacial torque favouring ordering along density gradients. We argue that our minimal model unifies various previous observations of chaotic behaviour in dry active matter into a general universality class. Recent studies of active matter, comprising particles that convert internal energy to relative motion-exerting force or torque dipoles on the surrounding medium as they do so-reveal that these systems generally function far from equilibrium and possess no passive analogues. 1 Instead, their microscopic models can sometimes be grouped into unique ''universality'' classes, and identifying the corresponding continuous equations is currently an area of active research. 1-6 For systems with orientational order (i.e., active liquid crystals), two important classes of models that emerged in the process are momentum-conserving (''wet'') incom-pressible systems 1,7 and non-momentum conserving (''dry'') compressible ones, 1,8-10 with the vast majority of work dedicated to the former class. Here we propose a new universality class of dry active systems defined by the following continuum equations ‡ @ t r ¼ r 2 1 32 r þ mr 2 þ @ i @ j p 48 þ wr h i Q ij À lr 2 Q kl Q kl ð Þ ; (1) @ t Q ij ¼ 4 r r cr À 1 À aQ kl Q kl þ kr 2 Q ij þ zD ij r À b 1 D ij Q kl Q kl ð ÞÀb 2 Q kl D ij Q kl ; (2) where a tensorial field Q ij quantifies the nematic (apolar) ordering of the active particles, and r is their density; i, j = {x, y} denote the two-dimensional Cartesian components, and D ij = q i q j À (1/2)d ij q k q k. We demonstrate that these equations arise naturally from a microscopic kinetic theory of model mixtures of microtubules (MTs) and molecular motors (MMs). 1,11 While wet incom-pressible active gels are generically unstable to orientational fluctuations ultimately resulting in ''active turbulence'', 7,12,13 we show that compressible dry MT-MM mixtures undergo seemingly similar chaotic dynamics, which we name dry active turbulence. The underlying mechanism is, however, completely different and we use eqn (1) and (2) to elucidate it: importantly, in our case concentration inhomogenenities play a central role. We note that similar dynamical patterns were observed, mainly at the level of a kinetic theory, for a very different physical system, a suspension of flocking self-propelled particles with nematic alignment. 8,14-16 We therefore argue that dry active turbulence unifies various previous observations of chaotic behaviour in nematically ordered microtubules and flocking self-propelled particles. Here, we consider the dynamics of pattern formation in model MT-MM mixtures that have actively been studied as biological and synthetic instances of active matter. 1,11,17-20 On the one hand, they incorporate the essential ingredients of the mitotic spindle, 21-23 on the other hand, they closely mirror the so-called ''hierarchical active matter'', which can be self-assembled in the lab from MTs and MMs, in the presence of polyethylene glycol. 24-26

Molecular Biology of the Cell, 2019

Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, ... more Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, whether actin filaments (F-actin) are required for or are even present in mitotic spindles has long been controversial. Here we have developed improved methods for simultaneously preserving F-actin and microtubules in fixed samples and exploited them to demonstrate that F-actin is indeed associated with mitotic spindles in intact Xenopus laevis embryonic epithelia. We also find that there is an "F-actin cycle," in which the distribution and organization of spindle F-actin changes over the course of the cell cycle. Live imaging using a probe for F-actin reveals that at least two pools of F-actin are associated with mitotic spindles: a relatively stable internal network of cables that moves in concert with and appears to be linked to spindles, and F-actin "fingers" that rapidly extend from the cell cortex toward the spindle and make transient contact with the spindle poles. We conclude that there is a robust endoplasmic F-actin network in normal vertebrate epithelial cells and that this network is also a component of mitotic spindles. More broadly, we conclude that there is far more internal F-actin in epithelial cells than is commonly believed.

Developmental Cell, 2019

Tight junctions contribute to epithelial barrier function by selectively regulating the quantity ... more Tight junctions contribute to epithelial barrier function
by selectively regulating the quantity and type
of molecules that cross the paracellular barrier.
Experimental approaches to evaluate the effectiveness
of tight junctions are typically global, tissuescale
measures. Here, we introduce Zinc-based
Ultrasensitive Microscopic Barrier Assay (ZnUMBA),
which we used in Xenopus laevis embryos to visualize
short-lived, local breaches in epithelial barrier
function. These breaches, or leaks, occur as cell
boundaries elongate, correspond to visible breaks
in the tight junction, and are followed by transient
localized Rho activation, or Rho flares. We discovered
that Rho flares restore barrier function by
driving concentration of tight junction proteins
through actin polymerization and ROCK-mediated
localized contraction of the cell boundary. We
conclude that Rho flares constitute a damage
control mechanism that reinstates barrier function
when tight junctions become locally compromised
because of normally occurring changes in cell shape
and tissue tension.

Cell Reports, 2019

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mamma... more NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.

iScience, 2018

SUMMARY Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure thei... more SUMMARY Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure their faithful inheritance. How only a single procentriole is produced on each mother centriole remains enigmatic. We propose the first mechanistic biophysical model for procentriole initiation which posits that interactions between kinase PLK4 and its activator-substrate STIL are central for procentriole initiation. The model recapitulates the transition from a uniform ''ring'' of PLK4 surrounding the mother centriole to a single PLK4 ''spot'' that initiates procentriole assembly. This symmetry breaking requires autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4 by phosphorylated STIL. We find that in situ degradation of active PLK4 cannot break symmetry. The model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes destabilizes the PLK4 ring and produces instead a single PLK4 spot. Weakening of competition by overexpression of PLK4 and STIL causes progressive addition of supernumerary procentrioles, as observed experimentally.

Phys. Rev. E, 2024

We explore a biomimetic model that simulates a cell, with the internal cytoplasm represented by a... more We explore a biomimetic model that simulates a cell, with the internal cytoplasm represented by a
two-dimensional circular domain and the external cortex by a surrounding ring, both modeled using FitzHugh-
Nagumo systems. The external ring is dynamically influenced by a pacemaker-driven wave originating from
the internal domain, leading to the emergence of three distinct dynamical states based on the varying strengths
of coupling. The range of dynamics observed includes phase patterning, the propagation of phase waves, and
interactions between traveling and phase waves. A simplified linear model effectively explains the mechanisms
behind the variety of phase patterns observed, providing insights into the complex interplay between a cell’s
internal and external environments.

Current Biology, 2024

A new analysis of cytokinetic furrow ingression in the Caenorhabditis elegans zygote at high spat... more A new analysis of cytokinetic furrow ingression in the Caenorhabditis elegans zygote at high spatiotemporal
resolution demonstrates that, rather than being a process of steady, spatially uniform constriction, furrow
ingression is modulated by complex contractile oscillations that move around the furrow, possibly in the
form of propagating waves.

Nature Reviews Molecular Cell Biology, 2024

The Rho GTPases-RHOA, RAC1 and CDC42-are small GTP binding proteins that regulate basic biologica... more The Rho GTPases-RHOA, RAC1 and CDC42-are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function.

Proc. Natl. Acad. Sci. USA, 2023

To initiate directed movement, cells must become polarized, establishing a protrusive leading edg... more To initiate directed movement, cells must become polarized, establishing a protrusive leading edge and a contractile trailing edge. This symmetry-breaking process involves reorganization of cytoskeleton and asymmetric distribution of regulatory molecules. However, what triggers and maintains this asymmetry during cell migration remains largely elusive. Here, we established a micropatterning-based 1D motility assay to investigate the molecular basis of symmetry breaking required for directed cell migration. We show that microtubule (MT) detyrosination drives cell polarization by directing kinesin-1-based transport of the adenomatous polyposis coli (APC) protein to cortical sites. This is essential for the formation of cell's leading edge during 1D and 3D cell migration. These data, combined with biophysical modeling, unveil a key role for MT detyrosination in the generation of a positive feedback loop linking MT dynamics and kinesin-1-based transport. Thus, symmetry breaking during cell polarization relies on a feedback loop driven by MT detyrosination that supports directed cell migration.

Journal of Cell Biology, 2022

Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskelet... more Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics.

Molecular Biology of the Cell, 2022

Interest in cortical excitability-the ability of the cell cortex to generate traveling waves of p... more Interest in cortical excitability-the ability of the cell cortex to generate traveling waves of protein activity-has grown considerably over the past 20 years. Attributing biological functions to cortical excitability requires an understanding of the natural behavior of excitable waves and the ability to accurately quantify wave properties. Here we have investigated and quantified the onset of cortical excitability in Xenopus laevis eggs and embryos and the changes in cortical excitability throughout early development. We found that cortical excitability begins to manifest shortly after egg activation. Further, we identified a close relationship between wave properties-such as wave frequency and amplitude-and cell cycle progression as well as cell size. Finally, we identified quantitative differences between cortical excitability in the cleavage furrow relative to nonfurrow cortical excitability and showed that these wave regimes are mutually exclusive.

Journal of Cell Biology, 2022

Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier func... more Epithelial cell-cell junctions remodel in response to mechanical stimuli to maintain barrier function. Previously, we found that local leaks in tight junctions (TJs) are rapidly repaired by local, transient RhoA activation, termed "Rho flares," but how Rho flares are regulated is unknown. Here, we discovered that intracellular calcium flashes and junction elongation are early events in the Rho flare pathway. Both laser-induced and naturally occurring TJ breaks lead to local calcium flashes at the site of leaks. Additionally, junction elongation induced by optogenetics increases Rho flare frequency, suggesting that Rho flares are mechanically triggered. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSCs) reduces the amplitude of calcium flashes and diminishes the sustained activation of Rho flares. MSC-dependent calcium influx is necessary to maintain global barrier function by regulating reinforcement of local TJ proteins via junction contraction. In all, we uncovered a novel role for MSC-dependent calcium flashes in TJ remodeling, allowing epithelial cells to repair local leaks induced by mechanical stimuli.

Current Biology, 2021

The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic ... more The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division.1,2 During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed “cortical excitability.”3, 4, 5, 6, 7 In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho.7,8 These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation.9 In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics.10 This reconstituted system spontaneously develops two distinct types of self-organized cortical dynamics: singular excitable Rho and F-actin waves, and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the excitable dynamics previously characterized in vivo.7 These findings directly support the long-standing speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics.

Molecular Biology of the Cell, 2021

Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microrid... more Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally elongated protrusions on mucosal epithelial cells, where they form evenly spaced, mazelike patterns that dynamically remodel by fission and fusion. To characterize how microridges form their highly ordered, subcellular patterns and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridge spacing and alignment became increasingly well ordered. Imaging F-actin and non-muscle myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked fission and fusion rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging allowed us to image individual NMII minifilaments in the apical cortex of cells in live animals, revealing that minifilaments are tethered to protrusions and often connect adjacent microridges. NMII minifilaments connecting the ends of two microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. These findings demonstrate that as cells mature, cortical NMII activity orchestrates a remodeling process that creates an increasingly orderly microridge arrangement.

Current Biology, 2021

As the interface between the cell and its environment, the cell cortex must be able to respond to... more As the interface between the cell and its environment, the cell cortex must be able to respond to a variety of external stimuli. This is made possible in part by cortical excitability, a behavior driven by coupled positive and negative feedback loops that generate propagating waves of actin assembly in the cell cortex. Cortical excitability is best known for promoting cell protrusion and allowing the interpretation of and response to chemoattractant gradients in migrating cells. It has recently become apparent, however, that cortical excitability is involved in the response of the cortex to internal signals from the cell-cycle regulatory machinery and the spindle during cell division. Two overlapping functions have been ascribed to cortical excitability in cell division: control of cell division plane placement, and amplification of the activity of the small GTPase Rho at the equatorial cortex during cytokinesis. Here, we propose that cortical excitability explains several important yet poorly understood features of signaling during cell division. We also consider the potential advantages that arise from the use of cortical excitability as a signaling mechanism to regulate cortical dynamics in cell division.

Journal of Cell Biology, 2021

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin fi... more The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.

Soft Matter, 2020

We study the dynamics of pattern formation in a minimal model for active mixtures made of microtu... more We study the dynamics of pattern formation in a minimal model for active mixtures made of microtubules and molecular motors. We monitor the evolution of the (conserved) microtubule density and of the (non-conserved) nematic order parameter, focusing on the effects of an ''anchoring'' term that provides a direct coupling between the preferred microtubule direction and their density gradient. The key control parameter is the ratio between activity and elasticity. When elasticity dominates, the interplay between activity and anchoring leads to formation of banded structures that can undergo additional bending, rotational or splaying instabilities. When activity dominates, the nature of anchoring instead gives rise to a range of active cellular solids, including aster-like networks, disordered foams and spindle-like patterns. We speculate that the introduced ''active model C'' with anchoring is a minimal model to describe pattern formation in a biomimetic analogue of the microtubule cytoskeleton.

Cells, 2020

Cellular morphogenesis is governed by the prepattern based on the symmetry-breaking emergence of ... more Cellular morphogenesis is governed by the prepattern based on the symmetry-breaking emergence of dense protein clusters. Thus, a cluster of active GTPase Cdc42 marks the site of nascent bud in the baker's yeast. An important biological question is which mechanisms control the number of pattern maxima (spots) and, thus, the number of nascent cellular structures. Distinct flavors of theoretical models seem to suggest different predictions. While the classical Turing scenario leads to an array of stably coexisting multiple structures, mass-conserved models predict formation of a single spot that emerges via the greedy competition between the pattern maxima for the common molecular resources. Both the outcome and the kinetics of this competition are of significant biological importance but remained poorly explored. Recent theoretical analyses largely addressed these questions, but their results have not yet been fully appreciated by the broad biological community. Keeping mathematical apparatus and jargon to the minimum, we review the main conclusions of these analyses with their biological implications in mind. Focusing on the specific example of pattern formation by small GTPases, we speculate on the features of the patterning mechanisms that bypass competition and favor formation of multiple coexisting structures and contrast them with those of the mechanisms that harness competition to form unique cellular structures.

Journal of Cell Biology, 2020

Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regul... more Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regulating their formation and arrangement are largely unknown. To study how protrusions form, we focused on the morphogenesis of microridges, elongated actin-based structures that are arranged in maze-like patterns on the apical surfaces of zebrafish skin cells. Microridges form by accreting simple finger-like precursors. Live imaging demonstrated that microridge morphogenesis is linked to apical constriction. A nonmuscle myosin II (NMII) reporter revealed pulsatile contractions of the actomyosin cortex, and inhibiting NMII blocked apical constriction and microridge formation. A biomechanical model suggested that contraction reduces surface tension to permit the fusion of precursors into microridges. Indeed, reducing surface tension with hyperosmolar media promoted microridge formation. In anisotropically stretched cells, microridges formed by precursor fusion along the stretch axis, which computational modeling explained as a consequence of stretch-induced cortical flow. Collectively, our results demonstrate how contraction within the 2D plane of the cortex can pattern 3D cell surfaces.

F1000 Research, 2019

Small GTPases are organizers of a plethora of cellular processes. The time and place of their act... more Small GTPases are organizers of a plethora of cellular processes. The time and place of their activation are tightly controlled by the localization and activation of their regulators, guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Remarkably, in some systems, the upstream regulators of GTPases are also found downstream of their activity. Resulting feedback loops can generate complex spatiotemporal dynamics of GTPases with important functional consequences. Here we discuss the concept of positive autoregulation of small GTPases by the GEF-effector feedback modules and survey recent developments in this exciting area of cell biology.

Soft Matter, 2019

We study the dynamics and phase behaviour of a dry suspension of microtubules and molecular motor... more We study the dynamics and phase behaviour of a dry suspension of microtubules and molecular motors. We obtain a set of continuum equations by rigorously coarse graining a microscopic model where motor-induced interactions lead to parallel or antiparallel ordering. Through numerical simulations, we show that this model generically creates either stable stripes, or a never-settling pattern where stripes periodically form, rotate and then split up. We derive a minimal model which displays the same instability as the full model, and clarifies the underlying physical mechanism. The necessary ingredients are an extensile flux arising from microtubule sliding and an interfacial torque favouring ordering along density gradients. We argue that our minimal model unifies various previous observations of chaotic behaviour in dry active matter into a general universality class. Recent studies of active matter, comprising particles that convert internal energy to relative motion-exerting force or torque dipoles on the surrounding medium as they do so-reveal that these systems generally function far from equilibrium and possess no passive analogues. 1 Instead, their microscopic models can sometimes be grouped into unique ''universality'' classes, and identifying the corresponding continuous equations is currently an area of active research. 1-6 For systems with orientational order (i.e., active liquid crystals), two important classes of models that emerged in the process are momentum-conserving (''wet'') incom-pressible systems 1,7 and non-momentum conserving (''dry'') compressible ones, 1,8-10 with the vast majority of work dedicated to the former class. Here we propose a new universality class of dry active systems defined by the following continuum equations ‡ @ t r ¼ r 2 1 32 r þ mr 2 þ @ i @ j p 48 þ wr h i Q ij À lr 2 Q kl Q kl ð Þ ; (1) @ t Q ij ¼ 4 r r cr À 1 À aQ kl Q kl þ kr 2 Q ij þ zD ij r À b 1 D ij Q kl Q kl ð ÞÀb 2 Q kl D ij Q kl ; (2) where a tensorial field Q ij quantifies the nematic (apolar) ordering of the active particles, and r is their density; i, j = {x, y} denote the two-dimensional Cartesian components, and D ij = q i q j À (1/2)d ij q k q k. We demonstrate that these equations arise naturally from a microscopic kinetic theory of model mixtures of microtubules (MTs) and molecular motors (MMs). 1,11 While wet incom-pressible active gels are generically unstable to orientational fluctuations ultimately resulting in ''active turbulence'', 7,12,13 we show that compressible dry MT-MM mixtures undergo seemingly similar chaotic dynamics, which we name dry active turbulence. The underlying mechanism is, however, completely different and we use eqn (1) and (2) to elucidate it: importantly, in our case concentration inhomogenenities play a central role. We note that similar dynamical patterns were observed, mainly at the level of a kinetic theory, for a very different physical system, a suspension of flocking self-propelled particles with nematic alignment. 8,14-16 We therefore argue that dry active turbulence unifies various previous observations of chaotic behaviour in nematically ordered microtubules and flocking self-propelled particles. Here, we consider the dynamics of pattern formation in model MT-MM mixtures that have actively been studied as biological and synthetic instances of active matter. 1,11,17-20 On the one hand, they incorporate the essential ingredients of the mitotic spindle, 21-23 on the other hand, they closely mirror the so-called ''hierarchical active matter'', which can be self-assembled in the lab from MTs and MMs, in the presence of polyethylene glycol. 24-26

Molecular Biology of the Cell, 2019

Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, ... more Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, whether actin filaments (F-actin) are required for or are even present in mitotic spindles has long been controversial. Here we have developed improved methods for simultaneously preserving F-actin and microtubules in fixed samples and exploited them to demonstrate that F-actin is indeed associated with mitotic spindles in intact Xenopus laevis embryonic epithelia. We also find that there is an "F-actin cycle," in which the distribution and organization of spindle F-actin changes over the course of the cell cycle. Live imaging using a probe for F-actin reveals that at least two pools of F-actin are associated with mitotic spindles: a relatively stable internal network of cables that moves in concert with and appears to be linked to spindles, and F-actin "fingers" that rapidly extend from the cell cortex toward the spindle and make transient contact with the spindle poles. We conclude that there is a robust endoplasmic F-actin network in normal vertebrate epithelial cells and that this network is also a component of mitotic spindles. More broadly, we conclude that there is far more internal F-actin in epithelial cells than is commonly believed.

Developmental Cell, 2019

Tight junctions contribute to epithelial barrier function by selectively regulating the quantity ... more Tight junctions contribute to epithelial barrier function
by selectively regulating the quantity and type
of molecules that cross the paracellular barrier.
Experimental approaches to evaluate the effectiveness
of tight junctions are typically global, tissuescale
measures. Here, we introduce Zinc-based
Ultrasensitive Microscopic Barrier Assay (ZnUMBA),
which we used in Xenopus laevis embryos to visualize
short-lived, local breaches in epithelial barrier
function. These breaches, or leaks, occur as cell
boundaries elongate, correspond to visible breaks
in the tight junction, and are followed by transient
localized Rho activation, or Rho flares. We discovered
that Rho flares restore barrier function by
driving concentration of tight junction proteins
through actin polymerization and ROCK-mediated
localized contraction of the cell boundary. We
conclude that Rho flares constitute a damage
control mechanism that reinstates barrier function
when tight junctions become locally compromised
because of normally occurring changes in cell shape
and tissue tension.

Cell Reports, 2019

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mamma... more NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.

iScience, 2018

SUMMARY Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure thei... more SUMMARY Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure their faithful inheritance. How only a single procentriole is produced on each mother centriole remains enigmatic. We propose the first mechanistic biophysical model for procentriole initiation which posits that interactions between kinase PLK4 and its activator-substrate STIL are central for procentriole initiation. The model recapitulates the transition from a uniform ''ring'' of PLK4 surrounding the mother centriole to a single PLK4 ''spot'' that initiates procentriole assembly. This symmetry breaking requires autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4 by phosphorylated STIL. We find that in situ degradation of active PLK4 cannot break symmetry. The model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes destabilizes the PLK4 ring and produces instead a single PLK4 spot. Weakening of competition by overexpression of PLK4 and STIL causes progressive addition of supernumerary procentrioles, as observed experimentally.