Actin polymerization and crosslinking drive left-right asymmetry in single cell and cell collectives

Abstract

Deviations from mirror symmetry in the development of bilateral organisms are highly stereotypic and genetically predetermined, but their mechanisms are not sufficiently understood. At the cellular level, self-organization of the actin cytoskeleton results in chiral actin swirling, and cells in groups confined to micropatterns demonstrate chiral cell alignment. The relationship between individual and collective cell chirality is unclear, and molecular players involved remain essentially unidentified. Here, by screening major actin-associated proteins and deep-learning-based morphometric analysis of actin patterns, we found that knockdowns of specific actin polymerization regulators, such as mDia1, ARPC2, and cofilins 1&2, abolished chiral actin swirling, while depletion of profilin 1 and CapZβ, reversed its direction in an actin crosslinker α-actinin1-dependent manner. Analysis of these and other knockdowns and pharmacological treatments revealed a robust correlation between their effects on the chirality of individual cells and confined cell groups. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry.One Sentence SummaryCell chirality determined by specific regulators of actin polymerization drives left-right asymmetry emergence in cell groups

Cell Chirality as a Novel Measure for Cytotoxicity

“…[53,54] Cell chirality seems to be built upon the functionalities of the actin cytoskeleton, as repeatedly demonstrated by multiple studies at different levels in the past decade. [40][41][42][43][44][45][46][47][48][49][50][51][52][55][56][57][58][59] However, the exact connection between chirality at a multicellular level and molecular chirality remains elusive. The characterization and quantification of cell chirality in in vitro experimental settings are critical for the systematic investigation of the implications of this novel cellular property in development and disease.…”

“…To this end, various engineering techniques have been developed in the past and demonstrated in different applications. [40][41][42][43][44][45][46][47][48][49][50][51] Currently, chirality characterization approaches are mostly imaging-based assays in both 2D and 3D, including the micropatterning-based method, [40][41][42][43][44][45][46]55,57,58,[61][62][63] the organelle positioning method, [44,45,50,51] 3D Matrigel bilayer method, [47,48] and cell motility-based methods. [60] In this section, we will comprehensively review the features and mechanisms of currently available chirality characterization methods and outlines their advantages and disadvantages for biomedical applications (Table 1).…”

“…By patterning individual cells on circular islands, this method can potentially avoid the influence of cell morphologies and allow for automatic analyses. Additionally, in a recent work, [58] a deep-learning computational algorithm was developed to quantitatively characterize the radial actin patterns by segmenting fibers into concentric rings and calculated average fiber tilting. This development increased the efficiency and accuracy of this in vitro assay.…”

“…Despite their uniqueness and potential advantages, these chirality assays have several potential limitations. First, because most chirality assays are micropatterning-based techniques, [40][41][42][43][44][45][46]57,58,[61][62][63] the integration of microcontact printing into other biomimetic systems such as microfluidic platforms can present significant technical difficulties in terms of device fabrication and batch quality control. Although methods like organelle positioning [44,45,50,51] can be used as an alternative in certain cases, live imaging capability will be limited as in situ immunofluorescent labeling of multiple organelles will be required.…”

Cell chirality reversal through tilted balance between polymerization of radial fibers and clockwise-swirling of transverse arc

“…However, it has been intriguing and puzzling of how single form of molecular chirality can be manifested in diverse chirality at cellular level that is even mirrored from one cell type to another 12,17 . Recently, righthanded axial spinning of radial ber during polymerization has been proposed as the underlying mechanism for the anticlockwise (ACW) chiral pattern of actin lament 25 . In addition, such ACW chirality can be reversed through overexpression of αactinin-1 that cross-links the actin lament or modulation of actin polymerization and depolymerization 19,25−27 .…”