Search Results (8)

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell’s microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed. Full article

Metastasis, in which cancer cells migrate to other tissues and form new tumors, is a major cause of both cancer death and treatment failure. In a previous study, benproperine (Benp) was identified as a cancer cell migration inhibitor and an inhibitor of actin-related protein 2/3 complex subunit 2 (ARPC2). However, Benp is a racemic mixture, and which stereoisomer is the active isomer remains unclear. In this study, we found that S-Benp is an active isomer and inhibits the migration and invasion of cancer cells much more strongly than R-Benp, with no effect on normal cells. The metastasis inhibitory effect of S-Benp was also verified in an animal model. Validating that inhibitors bind to their targets in cells and tissues has been a very challenging task in drug discovery. The direct interactions between ARPC2 and S-Benp were verified by surface plasmon resonance analysis (SPR), a cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS). In the mutant study with ARPC2^F225A cells, S-Benp did not bind to ARPC2^F225A according to CETSA and DARTS. Furthermore, we validated that S-Benp colocalized with ARPC2 in cancer cells and directly bound to ARPC2 in tumor tissues using Cy3-conjugated S-Benp according to CETSA. Finally, actin polymerization assays and immunocytochemistry showed that S-Benp suppressed actin remodeling such as lamellipodium formation. Taken together, our data suggest that S-Benp is an active stereoisomer of Benp and a potential metastasis inhibitor via ARPC2 binding. Full article

(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment. Full article

Muscle-invasive bladder cancer (MIBC) leads to a large societal burden. Recently, tRNA-derived small RNAs (tsRNAs), a novel type of noncoding RNA (ncRNAs), have been identified. However, the expression patterns and functions of tsRNAs in MIBC have not yet been identified. Here, RNA sequencing, bioinformatics, and quantitative reverse transcription- polymerase chain reaction (qRT-PCR) were used to screen the expression profiles and predict the potential roles of tsRNAs in MIBC. Of 406 tsRNAs differentially expressed in MIBC tissues, 91 tsRNAs were significantly differentially expressed. Then, four candidate tsRNAs, tiRNA-1:34-Val-CAC-2, tiRNA-1:33-Gly-GCC-1, tRF-1:32-Gly-GCC-1, and tRF-+1:T20-Ser-TGA-1, were selected. Next, a bioinformatics analysis showed the potential target genes and tsRNA–mRNA network. The most significant and meaningful terms of gene ontology were the positive regulation of the phosphate metabolic process, lamellipodium, and protein-cysteine S-acyltransferase activity in the biological process, cellular component, and molecular function, respectively. In addition, the top four pathways were predicted by the Kyoto Encyclopedia of Genes and Genomes database (KEGG). Finally, qRT-PCR demonstrated a similar expression pattern compared to sequencing data for the candidate tsRNAs. In short, we find differential expression profiles and predict that tiRNA-1:33-Gly-GCC-1, tRF-1:32-Gly-GCC-1, and tRF-+1:T20-Ser-TGA-1 are very likely to engage in the pathophysiological process of MIBC via regulating the target genes in the key pathways. Full article

Vimentin, an intermediate filament protein present in leukocytes, blood vessel endothelial cells, and multiple mesenchymal cells, such as mouse embryonic fibroblasts (MEF 3T3), is crucial for various cellular processes, as well as for maintaining the integrity and durability (stability) of the cell cytoskeleton. Vimentin intermediate filaments (VIFs) adhere tightly to the nucleus and spread to the lamellipodium and tail of the cell, serving as a connector between the nucleus, and the cell’s edges, especially in terms of transferring mechanical signals throughout the cell. How these signals are transmitted exactly remains under investigation. In the presented work, we propose that vimentin is involved in that transition by influencing the shape of the nucleus through the formation of nuclear blebs and grooves, as demonstrated by microscopic observations of healthy MEF (3T3) cells. Grooved, or “coffee beans” nuclei, have, to date, been noticed in several healthy cells; however, these structures are especially frequent in cancer cells—they serve as a significant marker for recognition of multiple cancers. We observed 288 MEF3T3 cells cultured on polyhydroxyoctanoate (PHO), polylactide (PLA), and glass, and we identified grooves, coaligned with vimentin fibers in the nuclei of 47% of cells cultured on PHO, 50% of cells on glass, and 59% of cells growing on PLA. We also observed nuclear blebs and associated their occurrence with the type of substrate used for cell culture. We propose that the higher rate of blebs in the nuclei of cells, cultured on PLA, is related to the microenvironmental features of the substrate, pH in particular. Full article

Changes in mechanical stimuli and the physiological environment are sensed by the cell. Thesechanges influence the cell’s motility patterns. The cell’s directional migration is dependent on the substrate stiffness. To describe such behavior of a cell, a tensegrity model was used. Cells with an extended lamellipodium were modeled. The internal elastic strain energy of a cell attached to the substrates with different stiffnesses was evaluated. The obtained results show that on the stiffer substrate, the elastic strain energy of the cell adherent to this substrate decreases. Therefore, the substrate stiffness is one of the parameters that govern the cell’s directional movement. Full article

Cells migrating over 2D substrates are required to polymerise actin at the leading edge to form lamellipodia protrusions and nascent adhesions to anchor the protrusion to the substrate. The major actin nucleator in lamellipodia formation is the Arp2/3 complex, which is activated by the WAVE regulatory complex (WRC). Using inducible Nckap1 floxed mouse embryonic fibroblasts (MEFs), we confirm that the WRC is required for lamellipodia formation, and importantly, for generating the retrograde flow of actin from the leading cell edge. The loss of NCKAP1 also affects cell spreading and focal adhesion dynamics. In the absence of lamellipodium, cells can become elongated and move with a single thin pseudopod, which appears devoid of N-WASP. This phenotype was more prevalent on collagen than fibronectin, where we observed an increase in migratory speed. Thus, 2D cell migration on collagen is less dependent on branched actin. Full article

Actin remodeling is frequently regulated by antagonistic activities driving protrusion and contraction downstream of Rac and Rho small GTPases, respectively. WAVE regulatory complex (WRC), which primarily operates downstream of Rac, plays pivotal roles in neuronal morphogenesis. Recently, two independent studies described de novo mutations in the CYFIP2 subunit of WRC, which caused intellectual disability (ID) in humans. Although mutations had been proposed to effect WRC activation, no experimental evidence for this was provided. Here, we made use of CRISPR/Cas9-engineered B16-F1 cell lines that were reconstituted with ID-causing CYFIP variants in different experimental contexts. Almost all CYFIP2-derived mutations (7 out of 8) promoted WRC activation, but to variable extent and with at least two independent mechanisms. The majority of mutations occurs in a conserved WAVE-binding region, required for WRC transinhibition. One mutation is positioned closely adjacent to the Rac-binding A site and appears to ease Rac-mediated WRC activation. As opposed to these gain-of-function mutations, a truncating mutant represented a loss-of-function variant and failed to interact with WRC components. Collectively, our data show that explored CYFIP2 mutations frequently, but not always, coincide with WRC activation and suggest that normal brain development requires a delicate and precisely tuned balance of neuronal WRC activity. Full article