PLEKHA5 regulates mitotic progression by promoting APC/C localization to microtubules

The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction. A microtubule-localized proximity biotinylation tool revealed that depletion of PLEKHA5 decreased the extent of APC/C association with microtubules. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

Push or pull: how cytoskeletal crosstalk facilitates nuclear movement through 3D environments

As cells move from two-dimensional (2D) surfaces into complex 3D environments, the nucleus becomes a barrier to movement due to its size and rigidity. Therefore, moving the nucleus is a key step in 3D cell migration. In this review, we discuss how coordination between cytoskeletal and nucleoskeletal networks is required to pull the nucleus forward through complex 3D spaces. We summarize recent migration models which utilize unique molecular crosstalk to drive nuclear migration through different 3D environments. In addition, we speculate about the role of proteins that indirectly crosslink cytoskeletal networks and the role of 3D focal adhesions and how these protein complexes may drive 3D nuclear migration.

Different Involvement of Band 3 in Red Cell Deformability and Osmotic Fragility—A Comparative GP.Mur Erythrocyte Study

GP.Mur is a clinically important red blood cell (RBC) phenotype in Southeast Asia. The molecular entity of GP.Mur is glycophorin B-A-B hybrid protein that promotes band 3 expression and band 3–AQP1 interaction, and alters the organization of band 3 complexes with Rh/RhAG complexes. GP.Mur+ RBCs are more resistant to osmotic stress. To explore whether GP.Mur+ RBCs could be structurally more resilient, we compared deformability and osmotic fragility of fresh RBCs from 145 adults without major illness (47% GP.Mur). We also evaluated potential impacts of cellular and lipid factors on RBC deformability and osmotic resistivity. Contrary to our anticipation, these two physical properties were independent from each other based on multivariate regression analyses. GP.Mur+ RBCs were less deformable than non-GP.Mur RBCs. We also unexpectedly found 25% microcytosis in GP.Mur+ female subjects (10/40). Both microcytosis and membrane cholesterol reduced deformability, but the latter was only observed in non-GP.Mur and not GP.Mur+ normocytes. The osmotic fragility of erythrocytes was not affected by microcytosis; instead, larger mean corpuscular volume (MCV) increased the chances of hypotonic burst. From comparison with GP.Mur+ RBCs, higher band 3 expression strengthened the structure of RBC membrane and submembranous cytoskeletal networks and thereby reduced cell deformability; stronger band 3–AQP1 interaction additionally supported osmotic resistance. Thus, red cell deformability and osmotic resistivity involve distinct structural–functional roles of band 3.

Persistent fluid flows defined by active matter boundaries

Biological systems control ambient fluids through the self-organization of active protein structures, including flagella, cilia, and cytoskeletal networks. Self-organization of protein components enables the control and modulation of fluid flow fields on micron scales, however, the physical principles underlying the organization and control of active-matter-driven fluid flows are poorly understood. Here, we use an optically-controlled active-matter system composed of microtubule filaments and light-switchable kinesin motor proteins to analyze the emergence of persistent flow fields. Using light, we form contractile microtubule networks of varying size and shape, and demonstrate that the geometry of microtubule flux at the corners of contracting microtubule networks predicts the architecture of fluid flow fields across network geometries through a simple point force model. Our work provides a foundation for programming microscopic fluid flows with controllable active matter and could enable the engineering of versatile and dynamic microfluidic devices.

Megakaryocyte Cytoskeletal Proteins in Platelet Biogenesis and Diseases

Thrombopoiesis governs the formation of blood platelets in bone marrow by converting megakaryocytes into long, branched proplatelets on which individual platelets are assembled. The megakaryocyte cytoskeleton responds to multiple microenvironmental cues, including chemical and mechanical stimuli, sustaining the platelet shedding. During the megakaryocyte's life cycle, cytoskeletal networks organize cell shape and content, connect them physically and biochemically to the bone marrow vascular niche, and enable the release of platelets into the bloodstream. While the basic building blocks of the cytoskeleton have been studied extensively, new sets of cytoskeleton regulators have emerged as critical components of the dynamic protein network that supports platelet production. Understanding how the interaction of individual molecules of the cytoskeleton governs megakaryocyte behavior is essential to improve knowledge of platelet biogenesis and develop new therapeutic strategies for inherited thrombocytopenias caused by alterations in the cytoskeletal genes.

Vimentin Cytoskeleton Architecture Analysis on Polylactide and Polyhydroxyoctanoate Substrates for Cell Culturing

Polylactide (PLA), widely used in bioengineering and medicine, gained popularity due to its biocompatibility and biodegradability. Natural origin and eco-friendly background encourage the search of novel materials with such features, such as polyhydroxyoctanoate (P(3HO)), a polyester of bacterial origin. Physicochemical features of both P(3HO) and PLA have an impact on cellular response 32, i.e., adhesion, migration, and cell morphology, based on the signaling and changes in the architecture of the three cytoskeletal networks: microfilaments (F-actin), microtubules, and intermediate filaments (IF). To investigate the role of IF in the cellular response to the substrate, we focused on vimentin intermediate filaments (VIFs), present in mouse embryonic fibroblast cells (MEF). VIFs maintain cell integrity and protect it from external mechanical stress, and also take part in the transmission of signals from the exterior of the cell to its inner organelles, which is under constant investigation. Physiochemical properties of a substrate have an impact on cells’ morphology, and thus on cytoskeleton network signaling and assembly. In this work, we show how PLA and P(3HO) crystallinity and hydrophilicity influence VIFs, and we identify that two different types of vimentin cytoskeleton architecture: network “classic” and “nutshell-like” are expressed by MEFs in different numbers of cells depending on substrate features.

α2β1 integrins spatially restrict Cdc42 activity to stabilise adherens junctions

Background Keratinocytes form the main protective barrier in the skin to separate the underlying tissue from the external environment. In order to maintain this barrier, keratinocytes form robust junctions between neighbouring cells as well as with the underlying extracellular matrix. Cell–cell adhesions are mediated primarily through cadherin receptors, whereas the integrin family of transmembrane receptors is predominantly associated with assembly of matrix adhesions. Integrins have been shown to also localise to cell–cell adhesions, but their role at these sites remains unclear. Results Here we show that α2β1 integrins are enriched at mature keratinocyte cell–cell adhesions, where they play a crucial role in organising cytoskeletal networks to stabilize adherens junctions. Loss of α2β1 integrin has significant functional phenotypes associated with cell–cell adhesion destabilisation, including increased proliferation, reduced migration and impaired barrier function. Mechanistically, we show that α2β1 integrins suppress activity of Src and Shp2 at cell–cell adhesions leading to enhanced Cdc42–GDI interactions and stabilisation of junctions between neighbouring epithelial cells. Conclusion Our data reveals a new role for α2β1 integrins in controlling integrity of epithelial cell–cell adhesions.