Graphene Enhances Actin Filament Assembly Kinetics and Modulates NIH-3T3 Fibroblast Cell Spreading

Actin plays critical roles in various cellular functions, including cell morphogenesis, differentiation, and movement. The assembly of actin monomers into double-helical filaments is regulated in surrounding microenvironments. Graphene is an attractive nanomaterial that has been used in various biomaterial applications, such as drug delivery cargo and scaffold for cells, due to its unique physical and chemical properties. Although several studies have shown the potential effects of graphene on actin at the cellular level, the direct influence of graphene on actin filament dynamics has not been studied. Here, we investigate the effects of graphene on actin assembly kinetics using spectroscopy and total internal reflection fluorescence microscopy. We demonstrate that graphene enhances the rates of actin filament growth in a concentration-dependent manner. Furthermore, cell morphology and spreading are modulated in mouse embryo fibroblast NIH-3T3 cultured on a graphene surface without significantly affecting cell viability. Taken together, these results suggest that graphene may have a direct impact on actin cytoskeleton remodeling.

Single-molecule imaging of IQGAP1 regulating actin filament dynamics

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet there remains a limited understanding of how IQGAP proteins directly influence actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to observe IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results demonstrate that full-length human IQGAP1 forms dimers that stably bind to actin filament sides and transiently cap barbed ends. These interactions organize filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as an actin regulator in vivo, and how it may be regulated in different biological settings. [Media: see text] [Media: see text] [Media: see text]

Single-molecule imaging of IQGAP1 regulating actin filament dynamics in real time

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet it has remained poorly understood how IQGAP proteins directly regulate actin filament dynamics. To close this gap, we used single-molecule and single-filament TIRF microscopy to directly visualize IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results show that full-length human IQGAP1 forms dimers that stably bind to filament sides and transiently cap barbed ends. These interactions organize actin filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as a direct actin regulator in vivo, and how it is deployed and regulated in different biological settings.

Filament dynamics driven by ATP hydrolysis modulates membrane binding of the bacterial actin MreB

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod- shaped bacteria. While the eukaryotic actin utilizes ATP hydrolysis to drive filament treadmilling, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we report mechanistic insights into the interplay between nucleotide-binding, ATP hydrolysis and membrane-binding of Spiroplasma citri MreB5 (ScMreB5). Antiparallel double protofilament assembly of ScMreB5WT with ATP, ADP or AMPPNP and an ATPase deficient mutant ScMreB5E134A demonstrate that the filaments assemble independent of ATP hydrolysis. However, capture of the filament dynamics revealed that efficient filament formation, bundling through lateral interactions and filament disassembly are affected in ScMreB5E134A. Hence, the catalytic glutamate (Glu134 in ScMreB5) plays a dual role - it functions as a switch by sensing the ATP-bound state for filament assembly, and by assisting hydrolysis for triggering disassembly. Glu134 mutation also exhibits an allosteric effect on membrane binding, as observed from the reduced liposome binding compared to that of the wild type. Thus, ATP hydrolysis can modulate filament length and bundling, and consequently the orientation of MreB filaments on the cell membrane depending on the curvature. Binding of ScMreB5 with liposomes is mediated by surface charge-based interactions, demonstrating paralog and organism specific features for MreB function. We conclude that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis has been repurposed for modulating curvature-dependent organization of filaments on the membrane.

Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

Cofilin: A Promising Protein Implicated in Cancer Metastasis and Apoptosis

Cofilin is an actin-binding protein that regulates filament dynamics and depolymerization. The over-expression of cofilin is observed in various cancers, cofilin promotes cancer metastasis by regulating cytoskeletal reorganization, lamellipodium formation and epithelial-to-mesenchymal transition. Clinical treatment of cancer regarding cofilin has been explored in aspects of tumor cells apoptosis and cofilin related miRNAs. This review addresses the structure and phosphorylation of cofilin and describes recent findings regarding the function of cofilin in regulating cancer metastasis and apoptosis in tumor cells.

A novel putative microtubule-associated protein is involved in arbuscule development during arbuscular mycorrhiza formation

The formation of arbuscular mycorrhizal (AM) symbiosis requires plant root host cells to undergo major structural and functional reprogramming in order to house the highly branched AM fungal structure for the reciprocal exchange of nutrients. These morphological modifications are associated with cytoskeleton remodelling. However, molecular bases and the role of microtubules (MTs) and actin filament dynamics during AM formation are largely unknown. In this study, the tomato tsb gene, belonging to a Solanaceae group of genes encoding MT-associated proteins for pollen development, was found to be highly expressed in root cells containing arbuscules. At earlier stages of mycorrhizal development, tsb overexpression enhanced the formation of highly developed and transcriptionally active arbuscules, while tsb silencing hampers the formation of mature arbuscules and represses arbuscule functionality. However, at later stages of mycorrhizal colonization, tsb OE roots accumulate fully developed transcriptionally inactive arbuscules, suggesting that the collapse and turnover of arbuscules might be impaired by TSB accumulation. Imaging analysis of the MT cytoskeleton in cortex root cells overexpressing tsb revealed that TSB is involved in MT-bundling. Taken together, our results provide unprecedented insights into the role of novel MT-associated protein in MT rearrangements throughout the different stages of the arbuscule life cycle.

Filament dynamics in planar chemical gardens

Modelling describes oscillatory dynamics of precipitate filaments; growth of an ensemble of filaments is a self-organized dispersion mechanism.