Microscopic studies on severing properties of actin-binding protein: its potential use in therapeutic treatment of actin-rich inclusions

Actin is an important unit of the cytoskeletal system, involved in many cellular processes including cell motility, signaling, and intracellular trafficking. Various studies have been undertaken to understand the regulatory mechanisms pertaining actin functions, especially the ones controlled by actin-binding proteins. However, not much has been explored about the molecular aspects of these proteins implicated in various diseases. In this study, we aimed to demonstrate the molecular properties of gelsolin, an actin-severing protein on the disassembly of the aggregation of actin-rich intracellular inclusions, Hirano body. We observed a decreasing tendency of actin aggregation by co-sedimentation assay and transmission electron microscopy in the presence of gelsolin. Therefore, we provide suggestive evidence for the use of actin-severing protein in novel therapeutic strategies for neurodegenerative conditions.

A Novel Toxoplasma Inner Membrane Complex Suture-Associated Protein Regulates Suture Protein Targeting and Colocalizes with Membrane Trafficking Machinery

The inner membrane complex (IMC) is a peripheral membrane and cytoskeletal system that is organized into intriguing rectangular plates at the periphery of the parasite. The IMC plates are delimited by an array of IMC suture proteins that are tethered to both the membrane and the cytoskeleton and are thought to provide structure to the organelle.

Microtubules enter centre stage for morphogenesis

Cell shape changes are key to observable changes at the tissue level during morphogenesis and organ formation. The major driver of cell shape changes in turn is the actin cytoskeleton, both in the form of protrusive linear or branched dynamic networks and in the form of contractile actomyosin. Over the last 20 years, actomyosin has emerged as the major cytoskeletal system that deforms cells in epithelial sheets during morphogenesis. By contrast, the second major cytoskeletal system, microtubules, have so far mostly been assumed to serve ‘house-keeping' functions, such as directed transport or cell division, during morphogenetic events. Here, I will reflect on a subset of studies over the last 10 years that have clearly shown a major direct role for the microtubule cytoskeleton in epithelial morphogenesis, suggesting that our focus will need to be widened to give more attention and credit to this cytoskeletal system in playing an active morphogenetic role. This article is part of a discussion meeting issue ‘Contemporary morphogenesis'.

Dictyostelium Cell Fixation: Two Simple Tricks

We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system.

Molecular underpinnings of cytoskeletal cross-talk

Cross-talk between the microtubule and actin networks has come under intense scrutiny following the realization that it is crucial for numerous essential processes, ranging from cytokinesis to cell migration. It is becoming increasingly clear that proteins long-considered highly specific for one or the other cytoskeletal system do, in fact, make use of both filament types. How this functional duality of “shared proteins” has evolved and how their coadaptation enables cross-talk at the molecular level remain largely unknown. We previously discovered that the mammalian adaptor protein melanophilin of the actin-associated myosin motor is one such “shared protein,” which also interacts with microtubules in vitro. In a hypothesis-driven in vitro and in silico approach, we turn to early and lower vertebrates and ask two fundamental questions. First, is the capability of interacting with microtubules and actin filaments unique to mammalian melanophilin or did it evolve over time? Second, what is the functional consequence of being able to interact with both filament types at the cellular level? We describe the emergence of a protein domain that confers the capability of interacting with both filament types onto melanophilin. Strikingly, our computational modeling demonstrates that the regulatory power of this domain on the microscopic scale alone is sufficient to recapitulate previously observed behavior of pigment organelles in amphibian melanophores. Collectively, our dissection provides a molecular framework for explaining the underpinnings of functional cross-talk and its potential to orchestrate the cell-wide redistribution of organelles on the cytoskeleton.

Septins Coordinate with Microtubules and Actin to Initiate Cell Morphogenesis

Many organs are formed by a process of branching morphogenesis, which begins with the formation of cytoplasmic extensions from the basal surface of polarized cells in an epithelial sheet. To study this process, we used a system of polarized epithelial spheroids, which emit cytoplasmic extensions in response to treatment with hepatocyte growth factor. We found that these extensions contain both actin and microtubules, but also septins, which are localized to microtubule bundles and appear to be important in maintaining microtubule organization. We found that these extensions are highly dynamic and form at a non-linear rate. We also demonstrated that the coordinated activity of microtubules, actin, and septins is necessary for the formation and dynamic behavior of extensions. Each cytoskeletal system plays a district role in this process, with microtubules enabling persistent growth of the extensions, actin enabling extension dynamics, and septins organizing microtubules in the extensions and supporting the extension formation. Together, our data offer insights into the dynamics of early morphogenic extensions and the distinct, but coordinated, roles of cytoskeleton in early morphogenesis.

Friend or Foe: The Role of the Cytoskeleton in Influenza A Virus Assembly

Influenza A Virus (IAV) is a respiratory virus that causes seasonal outbreaks annually and pandemics occasionally. The main targets of the virus are epithelial cells in the respiratory tract. Like many other viruses, IAV employs the host cell’s machinery to enter cells, synthesize new genomes and viral proteins, and assemble new virus particles. The cytoskeletal system is a major cellular machinery, which IAV exploits for its entry to and exit from the cell. However, in some cases, the cytoskeleton has a negative impact on efficient IAV growth. In this review, we highlight the role of cytoskeletal elements in cellular processes that are utilized by IAV in the host cell. We further provide an in-depth summary of the current literature on the roles the cytoskeleton plays in regulating specific steps during the assembly of progeny IAV particles.

Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria

Carboxysomes are protein-based bacterial organelles encapsulating key enzymes of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we identify a small protein, termed McdB that localizes to carboxysomes and drives emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly stimulates McdA ATPase activity and its release from DNA, driving carboxysome-dependent depletion of McdA locally on the nucleoid and promoting directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a previously unknown class of self-organizing proteins that utilize a Brownian-ratchet mechanism to position carboxysomes in cyanobacteria, rather than a cytoskeletal system. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria.

H+- and Na+-elicited swift changes of the microtubule system in the biflagellated green alga Chlamydomonas

The microtubule cytoskeletal system is integral to diverse cellular processes. Although microtubules are known for dynamic instability, the system is tightly controlled in typical interphase animal cells. In contrast, diverse evidence suggests that the system is mercurial in the unicellular fresh water green alga, Chlamydomonas, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by fluctuation of intracellular H+ and Na+. These results suggest disparate sensitivity of this vital yet delicate system in diverse organisms; and illuminate how pH may drive crucial cellular processes; how plants respond to, and perhaps sense stresses; and how many species could be susceptible to accelerated changes in global environments.