Globular (G)-actin, the actin monomer, assembles into polarized filaments that form networks that can provide structural support, generate force and organize the cell. Many of these structures are highly dynamic and to maintain them, the cell relies on a large reserve of monomers. Classically, the G-actin pool has been thought of as homogenous. However, recent work has shown that actin monomers can exist in distinct groups that can be targeted to specific networks, where they drive and modify filament assembly in ways that can have profound effects on cellular behavior. This Review focuses on the potential factors that could create functionally distinct pools of actin monomers in the cell, including differences between the actin isoforms and the regulation of G-actin by monomer binding proteins, such as profilin and thymosin β4. Owing to difficulties in studying and visualizing G-actin, our knowledge over the precise role that specific actin monomer pools play in regulating cellular actin dynamics remains incomplete. Here, we discuss some of these unanswered questions and also provide a summary of the methodologies currently available for the imaging of G-actin.

/pdf/reconsidering-an-active-role-for-g-actin-in-cytoskeletal-13kbp5yx39.pdf

Reconsidering an active role for G-actin in cytoskeletal regulation

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

Glycerophospholipids - Emerging players in neuronal dendrite branching and outgrowth.

Neurons of the CNS elaborate highly branched dendritic arbors that host numerous dendritic spines, which serve as the postsynaptic platform for most excitatory synapses. The actin cytoskeleton plays an important role in dendrite development and spine formation, but the underlying mechanisms remain incompletely understood. Tropomodulins (Tmods) are a family of actin-binding proteins that cap the slow-growing (pointed) end of actin filaments, thereby regulating the stability, length, and architecture of complex actin networks in diverse cell types. Three members of the Tmod family, Tmod1, Tmod2, and Tmod3 are expressed in the vertebrate CNS, but their function in neuronal development is largely unknown. In this study, we present evidence that Tmod1 and Tmod2 exhibit distinct roles in regulating spine development and dendritic arborization, respectively. Using rat hippocampal tissues from both sexes, we find that Tmod1 and Tmod2 are expressed with distinct developmental profiles: Tmod2 is expressed early during hippocampal development, whereas Tmod1 expression coincides with synaptogenesis. We then show that knockdown of Tmod2, but not Tmod1, severely impairs dendritic branching. Both Tmod1 and Tmod2 are localized to a distinct subspine region where they regulate local F-actin stability. However, the knockdown of Tmod1, but not Tmod2, disrupts spine morphogenesis and impairs synapse formation. Collectively, these findings demonstrate that regulation of the actin cytoskeleton by different members of the Tmod family plays an important role in distinct aspects of dendrite and spine development.SIGNIFICANCE STATEMENT The Tropomodulin family of molecules is best known for controlling the length and stability of actin myofilaments in skeletal muscles. While several Tropomodulin members are expressed in the brain, fundamental knowledge about their role in neuronal function is limited. In this study, we show the unique expression profile and subcellular distribution of Tmod1 and Tmod2 in hippocampal neurons. While both Tmod1 and Tmod2 regulate F-actin stability, we find that they exhibit isoform-specific roles in dendrite development and synapse formation: Tmod2 regulates dendritic arborization, whereas Tmod1 is required for spine development and synapse formation. These findings provide novel insight into the actin regulatory mechanisms underlying neuronal development, thereby shedding light on potential pathways disrupted in a number of neurological disorders.

https://www.jneurosci.org/content/jneuro/38/48/10271.full.pdf

Tropomodulin Isoform-Specific Regulation of Dendrite Development and Synapse Formation

Regulation of actin dynamics in dendritic spines: Nanostructure, molecular mobility, and signaling mechanisms.

Dendritic spines are small postsynaptic compartments of excitatory synapses in the vertebrate brain that are modified during learning, aging, and neurological disorders. The formation and modification of dendritic spines depend on rapid assembly and dynamic remodeling of the actin cytoskeleton in this highly compartmentalized space, but the precise mechanisms remain to be fully elucidated. In this study, we report that spatiotemporal enrichment of actin monomers (G-actin) in dendritic spines regulates spine development and plasticity. We first show that dendritic spines contain a locally enriched pool of G-actin that can be regulated by synaptic activity. We further find that this G-actin pool functions in spine development and its modification during synaptic plasticity. Mechanistically, the relatively immobile G-actin pool in spines depends on the phosphoinositide PI(3,4,5)P3 and involves the actin monomer-binding protein profilin. Together, our results have revealed a novel mechanism by which dynamic enrichment of G-actin in spines regulates the actin remodeling underlying synapse development and plasticity.

/pdf/phosphoinositide-dependent-enrichment-of-actin-monomers-in-41bjk88jzd.pdf

Phosphoinositide-dependent enrichment of actin monomers in dendritic spines regulates synapse development and plasticity.

Biomechanics of Endothelial Tubule Formation Differentially Modulated by Cerebral Cavernous Malformation Proteins.

The Rho family GTPases are molecular switches that regulate cytoskeletal dynamics and cell movement through a complex spatiotemporal organization of their activity. In Patiria miniata (starfish) oocytes under in vitro experimental conditions (with overexpressed Ect2, induced expression of Δ90 cyclin B, and roscovitine treatment), such activity generates multiple co-existing regions of coherent propagation of actin waves. Here we use computational modeling to investigate the development and properties of such wave domains. The model reveals that the formation of wave domains requires a balance between the activation and inhibition in the Rho signaling motif. Intriguingly, the development of the wave domains is preceded by a stage of low-activity quasi-static patterns, which may not be readily observed in experiments. Spatiotemporal patterns of this stage and the different paths of their destabilization define the behavior of the system in the later high-activity (observable) stage. Accounting for a strong intrinsic noise allowed us to achieve good quantitative agreement between simulated dynamics in different parameter regimes of the model and different wave dynamics in Patiria miniata and wild type Xenopus laevis (frog) data. For quantitative comparison of simulated and experimental results, we developed an automated method of wave domain detection, which revealed a sharp reversal in the process of pattern formation in starfish oocytes. Overall, our findings provide an insight into spatiotemporal regulation of complex and diverse but still computationally reproducible cell-level actin dynamics.

/pdf/spatiotemporal-development-of-coexisting-wave-domains-of-rho-1ca5sipbhv.pdf

Spatiotemporal development of coexisting wave domains of Rho activity in the cell cortex

Cells polarize their growth or movement in many different physiological contexts. A key driver of polarity is the Rho GTPase Cdc42, which when activated becomes clustered or concentrated at polar sites. Multiple models for polarity establishment have been proposed. All of them rely on positive feedback to reinforce regions of high Cdc42 activity. Positive feedback can lead to bistability, a scenario in which cells can exist in either a polarized or unpolarized state under identical external conditions. Determining if the signaling circuit that drives Cdc42 polarity is bistable would provide important information about the mechanism that underlies polarity establishment and insights into the design features required for proper cellular function. We studied polarity establishment during the mating response of yeast. Using microfluidics to precisely control the temporal profile of mating pheromone and live-cell imaging to monitor the polarity process in single living cells, we found that the polarity circuit of yeast shows hysteresis, a characteristic feature of bistable systems. Our analysis also revealed that cells exposed to high pheromone concentrations rapidly lose polarity following a precipitous removal of pheromone. We used a reaction-diffusion model for polarity establishment to demonstrate that delayed negative regulation is sufficient to explain our experimental results. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Bistability in the polarity circuit of yeast

Rho-GTPases are central regulators within a complex signaling network that controls the cytoskeletal organization and cell movement. This network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, and their numerous effectors that provide mutual regulation and feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling using a simulation model which couples GTPase signaling with cell morphodynamics to capture the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of the time-lapsed recordings of cell dynamics and GTPase activity. This approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data.

/pdf/spatiotemporal-coordination-of-rac1-and-cdc42-at-the-whole-16jbxrk4.pdf

Siarhei Hladyshau

Papers

Phosphoinositide-dependent enrichment of actin monomers in dendritic spines regulates synapse development and plasticity.

Biomechanics of Endothelial Tubule Formation Differentially Modulated by Cerebral Cavernous Malformation Proteins.

Spatiotemporal development of coexisting wave domains of Rho activity in the cell cortex

Bistability in the polarity circuit of yeast

Spatiotemporal Coordination of Rac1 and Cdc42 at the Whole Cell Level during Cell Ruffling