Existing actin filaments orient new filament growth to provide structural memory of filament alignment during cytokinesis

AbstractDuring cytokinesis, animal cells rapidly remodel the equatorial cortex to build an aligned array of actin filaments called the contractile ring. Local reorientation of filaments by equatorial contraction is thought to underlie the emergence of filament alignment during ring assembly. Here, combining single molecule analysis and modeling in one-cell C. elegans embryos, we show that filaments turnover is far too fast for reorientation of single filaments by equatorial contraction/cortex compression to explain the observed alignment, even if favorably oriented filaments are selectively stabilized. Instead, by tracking single Formin/CYK-1::GFP speckles to monitor local filament assembly, we identify a mechanism that we call filament-guided filament assembly (FGFA), in which existing filaments serve as templates to guide/orient the growth of new filaments. We show that FGFA sharply increases the effective lifetime of filament orientation, providing structural memory that allows slow equatorial contraction to build and maintain highly aligned filament arrays, despite rapid turnover of individual filaments.

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Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments.

The Journal of Cell Biology ◽

10.1083/jcb.128.1.61 ◽

1995 ◽

Vol 128 (1) ◽

pp. 61-70 ◽

Cited By ~ 82

Author(s):

D A Schafer ◽

C Hug ◽

J A Cooper

Keyword(s):

Monoclonal Antibody ◽

Actin Filament ◽

Actin Filaments ◽

Binding Activity ◽

Actin Binding ◽

Mutant Form ◽

Actin Binding Protein ◽

Filament Assembly ◽

Skeletal Myotubes ◽

Aligned Array

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin-binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.

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Actin kinetics shapes cortical network structure and mechanics

Science Advances ◽

10.1126/sciadv.1501337 ◽

2016 ◽

Vol 2 (4) ◽

pp. e1501337 ◽

Cited By ~ 70

Author(s):

Marco Fritzsche ◽

Christoph Erlenkämper ◽

Emad Moeendarbary ◽

Guillaume Charras ◽

Karsten Kruse

Keyword(s):

Single Molecule ◽

Actin Filaments ◽

Length Distribution ◽

Living Cells ◽

Stochastic Simulations ◽

Cortical Actin ◽

Animal Cells ◽

Cellular Mechanics ◽

Cellular Processes ◽

Actin Cortex

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

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Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1201 ◽

2009 ◽

Vol 20 (8) ◽

pp. 2160-2173 ◽

Cited By ~ 57

Author(s):

Colleen T. Skau ◽

Erin M. Neidt ◽

David R. Kovar

Keyword(s):

Fission Yeast ◽

Actin Filament ◽

Actin Filaments ◽

Actin Polymerization ◽

Actin Assembly ◽

Animal Cells ◽

Contractile Ring ◽

Direct Role ◽

Ring Assembly

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

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Molecular organization of cytokinesis node predicts the constriction rate of the contractile ring

The Journal of Cell Biology ◽

10.1083/jcb.202008032 ◽

2021 ◽

Vol 220 (3) ◽

Author(s):

Kimberly Bellingham-Johnstun ◽

Erica Casey Anders ◽

John Ravi ◽

Christina Bruinsma ◽

Caroline Laplante

Keyword(s):

Single Molecule ◽

Actin Filaments ◽

Molecular Organization ◽

Yeast Cells ◽

Live Cells ◽

Contractile Ring ◽

Ring Assembly ◽

Assembly Mechanism ◽

Excess Number ◽

Single Molecule Localization Microscopy

The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.

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Cortical flow aligns actin filaments to form a furrow

eLife ◽

10.7554/elife.17807 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 62

Author(s):

Anne-Cecile Reymann ◽

Fabio Staniscia ◽

Anna Erzberger ◽

Guillaume Salbreux ◽

Stephan W Grill

Keyword(s):

Actin Filaments ◽

Network Architecture ◽

Ring Formation ◽

Material Parameters ◽

C Elegans ◽

Physical Mechanisms ◽

Ring Assembly ◽

Quantitative Level ◽

Flow Alignment

Cytokinesis in eukaryotic cells is often accompanied by actomyosin cortical flow. Over 30 years ago, Borisy and White proposed that cortical flow converging upon the cell equator compresses the actomyosin network to mechanically align actin filaments. However, actin filaments also align via search-and-capture, and to what extent compression by flow or active alignment drive furrow formation remains unclear. Here, we quantify the dynamical organization of actin filaments at the onset of ring assembly in the C. elegans zygote, and provide a framework for determining emergent actomyosin material parameters by the use of active nematic gel theory. We characterize flow-alignment coupling, and verify at a quantitative level that compression by flow drives ring formation. Finally, we find that active alignment enhances but is not required for ring formation. Our work characterizes the physical mechanisms of actomyosin ring formation and highlights the role of flow as a central organizer of actomyosin network architecture.

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Assembly and architecture of precursor nodes during fission yeast cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.201008171 ◽

2011 ◽

Vol 192 (6) ◽

pp. 1005-1021 ◽

Cited By ~ 134

Author(s):

Damien Laporte ◽

Valerie C. Coffman ◽

I-Ju Lee ◽

Jian-Qiu Wu

Keyword(s):

Plasma Membrane ◽

Fission Yeast ◽

Actin Filaments ◽

Myosin Ii ◽

Stable Node ◽

Animal Cells ◽

Contractile Ring ◽

Cell Interior ◽

Ring Assembly ◽

Positional Marker

The contractile ring is essential for cytokinesis in most fungal and animal cells. In fission yeast, cytokinesis nodes are precursors of the contractile ring and mark the future cleavage site. However, their assembly and architecture have not been well described. We found that nodes are assembled stoichiometrically in a hierarchical order with two modules linked by the positional marker anillin Mid1. Mid1 first recruits Cdc4 and IQGAP Rng2 to form module I. Rng2 subsequently recruits the myosin-II subunits Myo2 and Rlc1. Mid1 then independently recruits the F-BAR protein Cdc15 to form module II. Mid1, Rng2, Cdc4, and Cdc15 are stable node components that accumulate close to the plasma membrane. Both modules recruit the formin Cdc12 to nucleate actin filaments. Myo2 heads point into the cell interior, where they efficiently capture actin filaments to condense nodes into the contractile ring. Collectively, our work characterizing the assembly and architecture of precursor nodes defines important steps and molecular players for contractile ring assembly.

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Flow-independent accumulation of motor-competent non-muscle myosin II in the contractile ring is essential for cytokinesis

10.1101/333286 ◽

2018 ◽

Cited By ~ 1

Author(s):

DS Osorio ◽

FY Chan ◽

J Saramago ◽

J Leite ◽

AM Silva ◽

...

Keyword(s):

Motor Activity ◽

Myosin Ii ◽

Minor Role ◽

Cortical Actin ◽

Animal Cells ◽

Contractile Ring ◽

Myosin Motor ◽

C Elegans ◽

Ring Assembly ◽

Muscle Myosin

AbstractCytokinesis in animal cells requires the assembly of a contractile actomyosin ring, whose subsequent constriction physically separates the two daughter cells. Non-muscle myosin II (myosin) is essential for cytokinesis, but the role of its motor activity remains poorly defined. Here, we examine cytokinesis in C. elegans one-cell embryos expressing myosin motor mutants generated by genome editing. Motor-dead myosin, which is capable of binding F-actin, does not support cytokinesis, and embryos co-expressing motor-dead and wild-type myosin are delayed in cytokinesis. Partially motor-impaired myosin also delays cytokinesis and renders contractile rings more sensitive to reduced myosin levels. Thus, myosin motor activity, rather than its ability to cross-link actin filaments, drives contractile ring assembly and constriction. We further demonstrate that myosin motor activity is required for long-range cortical actin flows, but that flows per se play a minor role in contractile ring assembly. Our results suggest that flow-independent recruitment of motor-competent myosin to the cell equator is both essential and rate-limiting for cytokinesis.

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