Small GTPase Research Articles

The small GTPase CDC42 promotes axon growth through actin filament polymerization and this growth is driven by axonal localization of the mRNA encoding the prenylated CDC42 isoform (Prenyl-Cdc42). Here, we show that axonal Prenyl-Cdc42 mRNA levels and the mRNA's translation are decreased by growth-inhibiting stimulation and increased by growth-promoting stimulation. In contrast, axonal RhoA mRNA transport and translation are increased by growth-inhibiting but unaffected by growth-promoting stimuli. Localized increase in KHSRP in response to growth inhibitory stimulation, through elevation of intracellular Ca2+, promotes decrease in axonal levels of Prenyl-Cdc42 mRNA. Distinct 3'UTR motifs regulate transport and axonal levels of Prenyl-Cdc42 mRNA. KHSRP protein binds to a Prenyl-Cdc42 mRNA motif within nt 801-875 and the mRNA is remarkably increased in axons of Khsrp-/- mice. Depletion of the mRNA from sciatic nerve indicates that the increased axonal Prenyl-CDC42 contributes to the accelerated nerve regeneration when neuronal KHSRP is depleted.

Smoking-associated laryngeal squamous cell carcinoma (LSCC) is characterized by high metastatic potential and poor prognosis. However, the underlying molecular mechanisms remain insufficiently understood. This study utilized single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to explore the heterogeneity of the tumor microenvironment in smoking-associated LSCC. Thirteen distinct cellular subpopulations within the tumor microenvironment are identified, with STC2 and ITGA5 emerging as smoking-associated prognostic markers. STC2 exhibited bifurcated differentiation within tumor epithelial cells, categorized as Tumor_C1 and Tumor_C2. The two subtypes are linked to vascular permeability and DNA replication pathways, respectively. Mechanistically, nicotine activated the JAK2/STAT3 signaling pathway through CHRNA5, resulting in direct STAT3 binding to the STC2 promoter and modulation of its transcription. STC2 subsequently upregulated TGFBI, which interacted with ITGA5 on endothelial cells, regulating vascular permeability and facilitating hematogenous dissemination of LSCC cells. Furthermore, STC2 knockdown altered F-actin cytoskeletal dynamics by modulating small GTPase signaling, impairing filopodia formation and epithelial polarity restoration. This study elucidates the tumor-endothelial interactions mediated by STC2 and ITGA5 in smoking-associated LSCC, emphasizing their roles in tumor progression and vascular permeability. These findings suggest potential prognostic biomarkers and therapeutic targets to improve the clinical management of smoking-associated LSCC.

RAC1 Directly Phosphorylates Both PKM2 and FBP1 to Promote Radioresistance in Hepatocellular Carcinoma.

Family with sequence similarity 134, member B (FAM134B), known for its role as an ER-phagy receptor, has been implicated in the promotion of hepatocellular carcinoma (HCC) progression through the activation of the AKT signaling pathway. However, the precise mechanism underlying FAM134B's activation of AKT signaling remains to be elucidated. This study aimed to investigate the interaction between FAM134B and DEAD-box helicase 3 X-linked (DDX3X) and its implications for HCC. We found that FAM134B interacts with DDX3X, preventing its proteasomal degradation by reducing K48-linked polyubiquitination and enhancing K63-linked polyubiquitination. This stabilization of DDX3X is crucial for AKT signaling activation, as DDX3X is known to promote the transcription of Rac Family Small GTPase 1 (Rac1), a key activator of the AKT pathway. Our results confirmed that FAM134B activates AKT signaling through the DDX3X-Rac1-AKT axis in HCC. Furthermore, we observed that DDX3X is upregulated in HCC and contributes to tumor progression. Interestingly, DDX3X not only activates AKT signaling but also increases FAM134B expression by enhancing its transcriptional activity, suggesting a positive feedback loop between these two proteins in HCC. Lastly, we explored the therapeutic potential of combining the DDX3X inhibitor RK-33 with FAM134B knockdown in HCC treatment. Our findings indicate that this synergistic approach may offer a promising strategy for HCC therapy.

G1 to S phase transition protein (GSPT1), a small GTPase involved in translation termination, which promotes the progression of cancer cells, has emerged as an attractive potential therapeutic target for cancer treatment with the rapid breakthrough of molecular glue degraders (MGDs). Although the precise mechanism of GSPT1 in cancer biology is partially understood, in this review, the characteristics of GSPT1 expression and regulatory networks are systematically attempted to be addressed, from insights into the structure, expression, and molecular mechanisms, highlighting the distribution and isoform-specific signaling of GSPT1 in tumors. The clinical significance is emphasized, immune interactions, and oncogenic pathways of GSPT1-targeted therapies, proposing strategies to address current challenges and provide therapeutic opportunities for the application of GSPT1 degraders in precision oncology. A novel future direction is hoped to provide to enhance the treatment response of GSPT1 MGDs in clinical implications.

Layilin, an understudied C-type lectin receptor for hyaluronan, was initially hypothesized to regulate cell motility due to its binding partner, talin. Subsequent studies identified layilin as a receptor for hyaluronan with roles in regulating cell motility through interactions with key regulatory molecules upstream of cytoskeletal rearrangement: radixin, merlin, focal adhesion kinase (FAK), F-actin, and small GTPases such as RAC1, RAP1, and RhoA. Layilin is also associated with cell-cell interactions, co-localizing with integrins in both T-cells and platelets contributing to epithelial cell junction integrity. Recent studies have found that layilin also plays a role in inflammation, dependent on tissue and disease. In the context of cancer, multiple cancer cell types displaying increased layilin expression contributes to enhanced metastasis. Exhausted CD8+ T cells residing in the tumors exhibit high expression of layilin, with the receptor contributing to increased tissue anchoring and co-expressing with immune checkpoint resistance markers. In other contexts, such as inflammatory bowel disease and atherosclerosis, reduction of layilin results in worsened disease and inflammation. Transcriptomic and epigenetic studies have explored layilin as a prognostic marker, as layilin expression is elevated in multiple cancers, deep vein thrombosis, diabetes, and Alzheimer's. However, the mechanistic role of layilin in most of these studies remains unexplored. This review outlines current insights into Layilin as a molecular hub that links hyaluronan signaling with integrin activity and cytoskeletal dynamics, highlighting its roles in homeostasis, pathogenesis, disease prognosis, and therapeutic intervention across diverse conditions.

Background: Right ventricular failure (RVF) has a high mortality risk across multiple heart diseases, has no proven therapies, and has few identified candidate targets suitable for testing in preclinical models. Research Goal: Identify evolutionarily conserved signatures of adaptive and maladaptive RV responses in human dilated cardiomyopathy (DCM) and mouse pulmonary artery band (PAB). Methods: Total and phosphoproteomics was performed for 56 human RVs (n= 16 nonfailing (NF), n=40 DCM) and 18 mice (n=6 sham, n=12 PAB). We used hemodynamic and echocardiographic assessments of DCM and PAB RVs to differentiate disease-associated from adaptive and maladaptive signatures by Voom/Limma, weighted correlation network analyses, and direct queries for sex-conserved patterns. We performed pathway enrichment analyses in Enrichr. Results: Right ventricular DCM and PAB differential protein abundances were broadly but modestly conserved (rho 0.34, P<0.0001) and revealed loss of mitochondrial/metabolic proteins and increase in extracellular matrix/TGFβ, collagen, and cell junction proteins. Adaptive/maladaptive signatures converged on actin and cell membrane remodeling, metabolic, proteostatic, and fibrotic proteins. Adaptive responses included increase in skeletal muscle ACTA1, enhanced sarcomeric actin treadmilling (e.g. CFL2), and cell membrane repair mechanisms (e.g. PARVA and TRIM72 phosphorylation). Whereas non-sarcomeric actin remodeling was maladaptive driven by a noncanonical WNT signaling axis involving small GTPases, WAVE2 complex, and ARP2/3. Loss/inhibition of oxidative phosphorylation, fatty acid oxidation, and malate-aspartate shuttle occurred in DCM/PAB independent of outcomes, whereas activation of pyruvate metabolism via downregulation of PDK1/4 and disinhibition of PDH1A was adaptive. Adaptive proteostatic signatures included slowed protein synthesis and enhanced folding, with mixed evidence for increase and decreased protein turnover involving autophagy, proteasome, and urea cycle. Maladaptive fibrotic signatures included stepwise increases in COL12A1, COL18A1, FN1, FMOD, LTBP2, and POSTN. Conclusion: Signatures of human adaptive RV remodeling that are conserved in mice—and therefore testable—include enhanced sarcomeric actin turnover, cell membrane repair, activation of pyruvate metabolism, and chaperone capacity exceeding protein synthetic needs.

Molecular mechanisms of dsRNA uptake and intracellular trafficking in the fat body of Locusta migratoria.

Interplay between Rac1/RhoA and actin waves in giant epithelial cells: experiment and theory.

Poplar PdRabG3f inhibits root elongation and increases salt tolerance by enhancing endogenous abscisic acid synthesis.

Design, synthesis and biological evaluation of (E)-4-(2-((1H-indol-5-yl) methylene) hydrazineyl)-5,6,7,8-tetrahydropyrido [4',3':4,5] thieno[2,3-d] pyrimidine derivatives as Arf1-GEFs inhibitors for the treatment of colon cancer.

How can a cell navigate its environment without any external cues? Since such cues are not always present in the environment, cells rely on internal machinery to explore their surroundings. Although Rho GTPases are known for orchestrating cell motility, the intrinsic Rho GTPase-effector mechanisms governing spontaneous migration remain incompletely understood. Here we show an imaging-based method that profiles protein-protein interactions (PPIs) through phase-separated condensates. By applying this method to hundreds of interaction profiles between Rho small GTPases and their effector proteins, we uncovered two intrinsic mechanisms governing cell migration. Formin-like protein (FMNL) determines the front of the cell by restricting Cdc42 activity, establishing front-rear polarity. In contrast, Rac1-ROCK-interaction-mediated arc stress fiber formation at the front inherently enables spontaneous directional changes and enhances cellular responses to external cues. Our findings elucidate the intricate roles of the Rho GTPase-effector ensemble that governs cell migration behavior, revealing an intrinsic program for efficient motility strategies.

The small GTPase KRAS is a key driver of carcinogenesis when mutated, and significant progress has been made in targeting KRASG12C and other oncogenic variants. Building on our previous work demonstrating the potential of nucleotide-based inhibitors with an acrylamide warhead to target KRASG13C, we designed and synthesized a library of nucleotide-based compounds with cyclic linkers to explore the effect of warhead orientation on reactivity toward Cys13. Using mass spectrometry, kinetic studies, and protein X-ray crystallography, we validated the binding and reactivity of these modulators. In addition, computational predictions of the conformational space of the linkers and warheads provided insights into their reactivity, which agreed well with the experimental data. These findings advance our understanding of the structure-reactivity relationship in these nucleotide-based KRAS inhibitors and will be the basis for further optimization.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-22145-5.

Cellular geometry is tightly associated with the function of a cell. During tumour progression, cancer cells undergo changes in phenotypes and biological behaviour with deformations in cellular morphology. However, whether the morphological diversity of cancer cells correlates with the cellular phenotype, and the underlying mechanism of morphology-related function in cancer cells is still unclear. Here, we simplified the cellular morphology by clustering cancer cells into three categories based on two-dimensional cellular morphological features. The silence of caveolin-1 (Cav-1), the primary constituent of membrane caveolae, reproduced the morphological evolutionary behaviour of cancer cells, which is similar to the epithelial-mesenchymal transition process. The attenuation of dorsal stress fibres, the assembly of focal adhesions and the disorder of transverse arc fibres and their regulatory signals are demonstrated as the main morphological evolutionary tools of cancer cells. Moreover, a modified vertex model theoretically reconfirmed the evolutionary process of cellular morphology. Small GTPases and focal adhesion kinase signalling were implicated in Cav-1 knockdown-induced cytoskeletal remodelling and focal adhesion assembly. Both invitro and invivo studies have demonstrated that Cav-1-dependent morphological changes are closely associated with the self-renewal capacity of breast cancer cells. Overall, our work highlights new insight into the morphological diversity and the correlation between cellular shape and phenotype of cancer cells, and provides evidence that Cav-1 could affect cancer cell properties such as self-renewal capacity through maintaining the morphological stability.

The cytokine CSF-1 and its tyrosine kinase-encoding receptor CSF1R are important for the development and proliferation/survival of most tissue macrophages. We recently identified TNF-α-induced protein 2 (TNFAIP2) as a unique cellular regulator of CSF1R activation: TNFAIP2 increased the response of macrophages to CSF-1, which might be explained at least in part by the induction of CSF1R clustering by TNFAIP2, since CSF1R clusters accelerate CSF-1-induced CSF1R dimerization/activation. Here, we report an enhanced trafficking of CSF1R to the cell surface as an additional mechanism by which TNFAIP2 increases macrophage response to CSF-1. The mutation in the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding site of TNFAIP2 or CSF1R, or the deprivation of cellular PIP2 reduced the enhanced CSF1R trafficking by TNFAIP2, indicating the importance of PIP2-mediated membrane localization of TNFAIP2 and CSF1R. Mechanistically, a small GTPase RalA and the exocyst complex involved in vesicle trafficking were required for the enhanced CSF1R trafficking by TNFAIP2. Interestingly, the RalA - exocyst complex cascade was also required for the enhanced CSF1R clustering by TNFAIP2. Our results suggest that TNFAIP2 enhances intracellular trafficking and cluster formation of CSF1R through PIP2, RalA, and the exocyst complex, thereby increasing macrophage response to CSF-1. Our results also suggest that TNFAIP2 regulates other receptor tyrosine kinases.

Macroautophagy, a major self-degradation pathway in eukaryotic cells, utilizes autophagosomes to transport self-material to lysosomes for degradation. While microtubular transport is crucial for the proper function of autophagy, the exact roles of factors responsible for positioning autophagosomes remain incompletely understood. In this study, we performed a loss-of-function genetic screen targeting genes potentially involved in microtubular motility. A genetic background that blocks autophagosome-lysosome fusions was used to accurately analyze autophagosome positioning. We discovered that pre-fusion autophagosomes move towards the non-centrosomal microtubule organizing center (ncMTOC) in Drosophila fat cells, which requires a dynein-dynactin complex. This process is regulated by the small GTPases Rab7 and Rab39 together with their adaptors: Epg5 and ema, respectively. The dynein-dependent movement of vesicles toward the nucleus/ncMTOC is essential for efficient autophagosomal fusions with lysosomes and subsequent degradation. Remarkably, altering the balance of kinesin and dynein motors changes the direction of autophagosome movement, indicating a competitive relationship where normally dynein-mediated transport prevails. Since pre-fusion lysosomes were positioned similarly to autophagosomes, it indicates that pre-fusion autophagosomes and lysosomes converge at the ncMTOC, which increases the efficiency of vesicle fusions.

Macroautophagy, a major self-degradation pathway in eukaryotic cells, utilizes autophagosomes to transport self-material to lysosomes for degradation. While microtubular transport is crucial for the proper function of autophagy, the exact roles of factors responsible for positioning autophagosomes remain incompletely understood. In this study, we performed a loss-of-function genetic screen targeting genes potentially involved in microtubular motility. A genetic background that blocks autophagosome-lysosome fusions was used to accurately analyze autophagosome positioning. We discovered that pre-fusion autophagosomes move towards the non-centrosomal microtubule organizing center (ncMTOC) in Drosophila fat cells, which requires a dynein-dynactin complex. This process is regulated by the small GTPases Rab7 and Rab39 together with their adaptors: Epg5 and ema, respectively. The dynein-dependent movement of vesicles toward the nucleus/ncMTOC is essential for efficient autophagosomal fusions with lysosomes and subsequent degradation. Remarkably, altering the balance of kinesin and dynein motors changes the direction of autophagosome movement, indicating a competitive relationship where normally dynein-mediated transport prevails. Since pre-fusion lysosomes were positioned similarly to autophagosomes, it indicates that pre-fusion autophagosomes and lysosomes converge at the ncMTOC, which increases the efficiency of vesicle fusions.

Small GTPases are critical regulators of cellular processes, such as cell migration, and comprise a family of over 167 proteins in the human genome. Importantly, the location-dependent regulation of small GTPase activity is integral to coordinating cellular signaling. Currently, there are no generalizable methods for directly controlling the activity of these signaling enzymes with subcellular precision. To address this issue, we introduce a modular, optogenetic platform for the spatial control of small GTPase activity within living cells, termed spLIT-small GTPases. This platform enabled spatially precise control of cytoskeletal dynamics such as filopodia formation (spLIT-Cdc42) and directed cell migration (spLIT-Rac1). Furthermore, a spLIT-RhoA system uncovered previously unreported long-range RhoA signaling in HeLa cells, resulting in bipolar membrane retraction. These results establish spLIT-small GTPases as a versatile platform for the direct, spatial control of small GTPase signaling and demonstrate the ability to uncover spatially defined aspects of small GTPase signaling.

Rac1 is a small GTPase that regulates cell proliferation, migration, and differentiation processes crucial for development. Mutations in specific guanine nucleotide exchange factors and GTPases that regulate Rac1 are associated with Adams-Oliver syndrome (AOS), a syndrome characterized by congenital scalp defects and limb truncations. Rac1 deletion in mouse embryonic limb ectoderm causes limb truncation. However, the etiology of Rac1-associated cranial defects is unknown. To investigate the cranial defects, we used Pdgfra-Cre to delete Rac1 in cranial mesenchyme. Rac1-KO mice died perinatally and lacked the apical calvarium and overlying dermis, resembling defects seen in severe AOS. In control embryos, α-smooth muscle actin (αSMA) expression was spatially restricted to the apical mesenchyme, suggesting mechanical interactions between the growing brain and the overlying mesenchyme. In Rac1-KO embryos, proliferation of apical mesenchyme, expression of αSMA, and its regulator, serum response factor (SRF), were reduced. Remarkably, Srf-KO with Pdgfra-Cre recapitulated the phenotype observed in Rac1-KO mice. Together, these data suggest a model where Rac1 and SRF maintain apical fibroblasts in a mechanoresponsive and proliferative state to complete cranial development.

The RAS family of small GTPases is among the most frequently mutated gene families in human cancer. In pancreatic ductal adenocarcinoma (PDAC), ∼95% of cases harbor an activating KRAS mutation, primarily at codon 12, 13, or 61, with G12D being the most common overall (40%). In contrast, the KRASQ61L mutation, though constitutively active, is virtually absent in tumors of patients with PDAC. This suggests that KRASQ61L may engage in distinct, allele-specific signaling that limits its ability to drive tumorigenesis. Determining the mechanisms that limit the occurrence of this mutation will aid in our understanding of the critical KRAS effectors and pathways that drive tumorigenesis. To investigate these mechanisms, we utilized a tightly controlled doxycycline-inducible KRAS expression system in an isogenic, immortalized pancreatic cell line, enabling direct comparison of KRASQ61L with the common PDAC mutant KRASG12D. Using TurboID proximity labeling alongside RNA sequencing, we mapped early effector interactions and transcriptional responses, revealing that KRASQ61L induces greater hyperactivation of the ERK/MAPK pathway, resulting in increased nuclear translocation of ERK1/2. Finally, pancreatic cells are highly tolerant to overexpression of KRASG12D, but KRASQ61L overexpression leads to impaired proliferation and increased apoptosis. These findings provide experimental support for the long-standing “Goldilocks” model of oncogenic signaling, in which too much ERK/MAPK pathway activation is detrimental to tumorigenesis. Our work offers a mechanistic explanation for the relative absence of KRASQ61L in PDAC and contributes to our understanding of KRAS allele–specific vulnerabilities, which can inform future therapeutic strategies targeting KRAS-driven pancreatic cancer.Significance:This study demonstrates that KRASQ61L drives hyperactivation of ERK/MAPK signaling and triggers apoptosis, which limits the proliferation of pancreatic cells. These findings support a “Goldilocks” model of RAS signaling and suggest that strong hyperactivation of the ERK/MAPK pathway contributes to the selective absence of KRASQ61L in pancreatic tumorigenesis.