Michael F. Olson

The actin cytoskeleton is a complex network of proteins that is constantly being remodelled and reorganized to orchestrate numerous essential cellular processes. From the regulation of proliferation, motility, cytokinesis through to signal transduction, and beyond, the dynamic nature of the actin cytoskeleton is vital to the enactment of these cellular functions. As a result, when there are aberrations in cytoskeleton dynamics, adverse consequences may be triggered. For example, dysregulation of the actin cytoskeleton has been linked to several neurodevelopmental and neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Additionally, actin cytoskeleton dysregulation has been shown to drive cancer growth and invasion, with altered expression levels of actin effectors and regulators being frequently associated with poorer prognoses and more invasive phenotypes. Given the complex and varied roles of the actin cytoskeleton and the ramifications of its dysregulation, the characterization of its key regulators is essential to understand disease etiology and to identify potential new therapeutic targets. This review highlights the interconnections of three protein families that are central to actin cytoskeleton modification, regulation, and function: the MICAL monooxygenase family, and the Rho and Rab GTPase families. The roles of the MICAL protein family and its functions in connecting Rho and Rab protein signalling will be discussed, with a particular focus on MICAL1.

Cell migration is critically important for development and for homeostatic processes such as wound healing, while its aberrant regulation may contribute to the development of pathological conditions and diseases. Efficient cell migration requires the coordination of many structural and signalling proteins ( e.g. Rho GTPases) that have key functions in controlling actin cytoskeleton organization and dynamics. Given that the actin cytoskeleton has a central role in orchestrating the complex series of events underlying cell migration, characterizing the actin-associated proteome can illuminate important molecular players that could be novel therapeutic targets for diseases associated with dysregulated cell migration. Here, we report a proteomic-based assay that uses a doxycycline-inducible proximity ligation assay to label actin-associated proteins (actin cytoskeletome) during the early stages of cell migration. We found that an unexpected actin-associated protein, Sec23b, influences the rate of cell migration by orchestrating the secretion and post-secretion maturation of Collagen I.

High-grade serous ovarian carcinoma (HGSOC) is characterized by high mortality rates and the frequent development of chemotherapy resistance. A hallmark of HGSOC progression is the formation of multicellular spheroids in malignant ascites that facilitate peritoneal dissemination and metastasis. While the CDC42-regulated kinases MRCKα and MRCKβ (MRCK) were previously found to be highly expressed in ovarian tumors and to be essential for cell migration and spheroid growth, the underlying molecular mechanisms were poorly defined. Mass spectrometry identified the RhoA-selective guanine nucleotide exchange factor GEF-H1 as a primary interacting partner of MRCKα. Functional assays revealed that MRCK inhibition or knockdown led to significantly increased levels of active GEF-H1 and subsequent RhoA activation. MRCKα was found to phosphorylate GEF-H1 on Ser174, and pharmacological inhibition of MRCK reduced this phosphorylation and decreased the association of GEF-H1 with α-Tubulin. Live-cell imaging and 3D assays demonstrated that MRCK inhibition disrupted cell-cell contacts and impaired the compaction of multicellular structures, ultimately reducing the viability of patient-derived organoids. These findings delineate a novel signaling crosstalk mechanism in which MRCKα represses GEF-H1-mediated RhoA activation to facilitate the formation of cell-cell contacts that contribute to the survival and growth of HGSOC cells in 3D multicellular structures. This study highlights MRCK as a potential therapeutic target to inhibit the growth and spread of HGSOC.

Phagocytosis of apoptotic cells maintains tissue homeostasis and regulates inflammation. A proposed facilitator of apoptotic cell clearance is the fragmentation of these cells into apoptotic bodies (ApoBDs) through cell-autonomous processes involving caspases and cytoskeletal rearrangement. Although this fragmentation process is considered a hallmark of apoptosis, its progression in tissue environments remains underexplored. Here, we examine the in vivo apoptotic dynamics of mouse thymocytes and pluripotent cells from zebrafish embryos. We show that the in vivo biogenesis of ApoBDs is independent of known cell-intrinsic regulators. Instead, fragmentation depends on actin-rich protrusions from neighboring resident phagocytes, which mechanically compress apoptotic cells to break them into smaller particles. Four-dimensional in vivo tracking of apoptotic cells reveals that both phagocyte-mediated fragmentation and phagocytosis are size sensitive, indicating that apoptotic size reduction mediated by phagocytes enhances their own clearance abilities. This non–cell-autonomous fragmentation ensures rapid apoptotic cell clearance, crucial for maintaining tissue homeostasis in physiological settings.

Accurate cell segmentation is an essential step in the quantitative analysis of fluorescence microscopy images. Pre-trained deep learning models for automatic cell segmentation such as those offered by Cellpose perform well across a variety of biological datasets but might still introduce segmentation errors. Although training custom models can improve accuracy, it often requires programming expertise and significant time, limiting the accessibility of automatic cell segmentation for many wet lab researchers. To address this gap, we developed ‘Toggle-Untoggle’, a standalone desktop application that enables intuitive, code-free quality control of automated cell segmentation. Our tool integrates the latest Cellpose ‘cyto3’ model, known for its robust performance across diverse cell types, while also supporting the ‘nuclei’ model and user-specified custom models to provide flexibility for a range of segmentation tasks. Through a user-friendly graphical interface, users can interactively toggle individual segmented cells on or off, merge or draw cell masks, and export morphological features and cell outlines for downstream analysis. Here, we demonstrate the utility of Toggle-Untoggle in enabling accurate, efficient single-cell analysis on real-world fluorescence microscopy data, with no coding skills required.

Apoptotic cells communicate to phagocytic cells through releasing soluble factors and apoptotic cell-derived extracellular vesicles. However, whether there are additional factors that remain attached at the site of cell death to signal to phagocytic cells is currently unknown. Here we show that apoptotic cell retraction generates a membrane-encased, F-actin-rich ‘footprint’ tightly anchored to the substrate that marks the site of cell death, coined ‘the FOotprint Of Death’ or FOOD. Formation of FOOD is observed frequently across many different cell types, apoptotic stimuli and surface composition. Mechanistically, FOOD formation is regulated by the protein kinase ROCK1. 3D time-lapse microscopy studies revealed that FOOD vesicularises into distinct large extracellular vesicles. These extracellular vesicles expose the ‘eat-me’ signal phosphatidylserine and can function to ‘flag’ the site of cell death to neighbouring phagocytes for efferocytosis. Under a viral infection setting, FOOD can harbour viral proteins and virions, and propagate infection to healthy cells. Together, this study has revealed another route of apoptotic cell-phagocyte communication.

Accurate cell segmentation is an essential step in the quantitative analysis of fluorescence microscopy images. Pre-trained deep learning models for automatic cell segmentation, such as Cellpose, offer strong performance across a variety of biological datasets but may still introduce segmentation errors. While training custom models can improve accuracy, it often requires programming expertise and significant time, limiting the accessibility of automatic cell segmentation for many wet lab researchers. To address this gap, we developed “Toggle-Untoggle”, a desktop application that combines automated segmentation using the Cellpose “cyto3” model with a user-friendly graphical interface for intuitive segmentation quality control. Our app allows users to refine results by interactively toggling individual segmented cells on or off without the need to manually edit segmentation masks, and to export morphological data and cell outlines for downstream analysis. Here we demonstrate the utility of “Toggle-Untoggle” in enabling accurate, efficient single-cell analysis on real-world fluorescence microscopy data, with no coding skills required.

Neuronal development is a highly regulated process that is dependent on the correct coordination of cellular responses to extracellular cues. In response to semaphorin axon guidance proteins, the MICAL1 protein is stimulated to produce reactive oxygen species that oxidize actin on specific methionine residues, leading to filamentous actin depolymerization and consequent changes in neuronal growth cone dynamics. Crossing genetically modified mice homozygous for floxed Mical1 (Mical1^fl/fl) alleles with transgenic mice expressing Cre recombinase under the control of a tyrosinase gene enhancer/promoter (Tyr::Cre) enabled conditional Mical1 deletion. Immunohistochemical analysis showed Mical1 expression in the cerebellum, which plays a prominent role in the coordination of motor movements, with reduced Mical1 expression in Mical1^fl/fl mice co‐expressing Tyr::Cre. Analysis of the gaits of mice running on a treadmill showed that both male and female Mical1^fl/fl, Tyr::Cre mutant mice had significant alterations to their striding patterns relative to wild‐type mice, although the specific aspects of their altered gaits differed between the sexes. Additional motor tests that involved movement on a rotating rod, descending a vertical pole, or crossing a balance beam did not show significant differences between the genotypes, suggesting that the effect of the Mical1^fl/fl, Tyr::Cre genetic modifications was only manifested during specific highly coordinated movements that contribute to running. These findings indicate that there is a behavioral consequence in Mical1^fl/fl, Tyr::Cre mutant mice that affects motor control as manifested by alterations in their gait.

Apoptosis is characterized by membrane blebbing and apoptotic body formation. Caspase cleavage of ROCK1 generates an active fragment that promotes actin–myosin-mediated contraction and membrane blebbing during apoptosis. Expression of caspase-resistant non-cleavable ROCK1 (Rock1 NC) prolonged survival of mice that rapidly develop B cell lymphomas due to Eµ-Myc transgene expression. Eµ-Myc; Rock1 NC mice had significantly fewer bone marrow cells relative to those in Eµ-Myc mice expressing wild-type ROCK1 (Rock1 WT), which was associated with altered cell cycle profiles. Circulating macrophage numbers were lower in Eµ-Myc; Rock1 NC mice, but there were higher levels of bone marrow macrophages, consistent with spontaneous cell death in Eµ-Myc; Rock1 NC mouse bone marrows being more inflammatory. Rock1 WT recipient mice transplanted with pre-neoplastic Eµ-Myc; Rock1 NC bone marrow cells survived longer than mice transplanted with Eµ-Myc; Rock1 WT cells, indicating that the survival benefit was intrinsic to the Eµ-Myc; Rock1 NC bone marrow cells. The results suggest that the apoptotic death of Eµ-Myc; Rock1 NC cells generates a proliferation-suppressive microenvironment in bone marrows that reduces cell numbers and prolongs B cell lymphoma mouse survival.