Biology subjects

Garcia-Mata, R.

Publications and source records attributed to Garcia-Mata, R..

8 recordsLinked to original sources

bioRxiv2026

A genetically encoded RhoG FRET biosensor reveals spatially compartmentalized RhoG-Rac1 signaling during cell protrusion

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Forster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor-stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

cell biology↗

bioRxiv2026

SGEF coordinates epithelial morphogenesis by regulating junction stability, collective migration, and extracellular matrix remodeling

Polarized epithelia are essential for organ function, and disruption of epithelial polarity is a hallmark of many diseases, including cancer. We previously showed that the RhoG-specific guanine nucleotide exchange factor SGEF interacts with the Scribble polarity complex to regulate epithelial junction assembly in 2D monolayers. However, its role in epithelial morphogenesis and lumen formation in 3D remains unknown. Here, we combined quantitative morphometric analysis with long-term live-cell imaging to investigate the role of SGEF during MDCK cyst development. SGEF KD disrupted normal lumenogenesis, producing enlarged cysts with multiple collapsed lumens accompanied by reduced E-cadherin, {beta}-catenin, and ZO-1 expression. Loss of SGEF also altered the distribution of the actomyosin network. Re-expression of WT SGEF restored the normal phenotype, whereas restoration of E-cadherin and ZO-1 partially rescued lumen architecture, identifying the loss of junction integrity as a key driver of the morphogenetic defects. Unexpectedly, live-cell imaging revealed increased motility and frequent cyst fusion in SGEF-KD cysts. Restoring E-cadherin levels abolished cyst migration, while inhibition of matrix metalloproteinases markedly restored normal cyst volume and lumen architecture, identifying extracellular matrix remodeling as an additional contributor to the SGEF-deficient phenotype. Together, these findings identify SGEF as a key regulator of epithelial morphogenesis, coordinating junction integrity, actomyosin organization, lumen formation, and collective migration.

cell biology↗

bioRxiv2025

Quantification of horizontal and vertical distribution of junctional proteins in fixed epithelial cells

Polarized epithelial cells form a tightly packed monolayer where individual cells are connected by cell-cell junctions, including tight junctions (TJ) and adherens junctions (AJ). Here, we present techniques for quantifying the horizontal and vertical distribution of junctional proteins in confluent, fixed epithelial cells. This approach is utilized to evaluate variations in the intensity and localization of the proteins that compose the AJ and TJ under different experimental conditions. Although our protocol is optimized for Madin-Darby Canine Kidney (MDCK) cells, it is adaptable to any cell line capable of forming cell-cell junctions. For complete details on the use and execution of this protocol, please refer to Rabino et al. (2024). 1 Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC="FIGDIR/small/651687v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1d1db4forg.highwire.dtl.DTLVardef@954ea1org.highwire.dtl.DTLVardef@5d43eborg.highwire.dtl.DTLVardef@1162077_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Critical-Size Defect Tibialis Anterior (TA) Muscle Regeneration using Ex-Vivo Mice Hindlimbs Culturing under Dynamic Mechanical Loading

In this study, we introduced an innovative computer-controlled ex vivo mice hindlimb culturing platform operating under dynamic loading, coupled with injectable cell-laden nanofibrous matrix (PNCOL), to investigate tissue response and therapeutic outcomes in critical size defect tibialis anterior (TA) muscle regeneration. The combination of mechanical stimulation and cell therapy offers a distinctive opportunity to delve into the regenerative rehabilitation field and create sustainable solutions in musculoskeletal (MSK) tissue regeneration. The application of mechanical loading on the whole mice hindlimbs increased total bone area and marrow area suggesting an increase in periosteal bone formation and resorption on the endosteal surface. Viability assessments confirmed the sustained culturing of the samples throughout the study. Then, the effect of mechanical loading and PNCOL injection on muscle regeneration at the TA defect site was evaluated. Histological analyses revealed enhanced muscle regeneration in PNCOL-treated hindlimbs. Structural analysis of the defect area through scanning electron microscopy (SEM) showed regeneration of ECM fibers at the defect site in PNCOL-treated groups. An analysis of cytokine levels in conditioned media at the end experiment showed changes in the number of proteins with the role in wound healing, muscle regeneration WNT, and IGF-1 signaling suggesting an anabolic effect of mechanical stimulation on muscle and bone. Similarly, gene expression analysis showed a significant upregulation of PAX7, Mrf4, MYF5, and TGF{beta}1 mRNA levels, indicating enhanced muscle regeneration after coupled mechanical loading and PNCOL treatments. Lastly, immunostaining showed an increase in tissue regeneration and anti-inflammatory response (CD206) in PNCOL-treated groups. Overall, the ex vivo hindlimb organ culturing platform- maintained tissue functions under mechanical loading, while PNCOL treatment promoted muscle tissue regeneration and reduced inflammation. These findings demonstrated the potential of multidimensional approaches for enhancing therapeutic outcomes in MSK disorders. In addition, this study aligns with the growing emphasis on minimizing the number of animals used in research and developing a robust sense of responsible animal experimentation through introducing dynamic ex-vivo muscle organ culturing platform.

bioengineering↗

bioRxiv2024

The Scribble/SGEF/Dlg1 complex regulates the stability of apical junctions in epithelial cells

SGEF, a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of the expression of both E-cadherin and ZO-1. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble/SGEF/Dlg1 complex. Our results show that an intact ternary complex is required to maintain the stability of the apical junctions, the expression of ZO-1, and TJ permeability. In contrast, only SGEF is necessary to regulate E-cadherin expression. The absence of SGEF destabilizes the E-cadherin/catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, {beta}-catenin signaling and the transcriptional repressor Slug.

cell biology↗

bioRxiv2024

A PACAP-activated network for secretion requires coordination of calcium influx and calcium mobilization

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLC{varepsilon}) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

cell biology↗

bioRxiv2022

ARHGAP17 regulates the spatiotemporal activity of Cdc42 at invadopodia

Invadopodia formation is regulated by Rho GTPases. However, the molecular mechanisms that control Rho GTPase signaling at invadopodia remain poorly understood. Here, we have identified ARHGAP17, a Cdc42-specific RhoGAP, as a key regulator of invadopodia in breast cancer cells and by RhoGAPs characterized a novel ARHGAP17-mediated signaling pathway that controls the spatiotemporal activity of Cdc42 during invadopodia turnover. Our results show that during invadopodia assembly, ARHGAP17 localizes to the invadopodia ring and restricts the activity of Cdc42 to the invadopodia core, where it promotes invadopodia growth. Invadopodia disassembly starts when ARHGAP17 translocates from the invadopodia ring to the core, in a process that is mediated by its interaction with the Cdc42 effector CIP4. Once at the core, ARHGAP17 inactivates Cdc42 to promote invadopodia disassembly. Our results in invadopodia provide new insights on the coordinated transition between the activation and inactivation of Rho GTPases.

cell biology↗

bioRxiv2020

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26s role in Salmonella epithelial cell invasion, identified serovar specific interactions, implicated ARHGEF26 in SopE-mediated invasion, and revealed that the ARHGEF26-associated proteins DLG1 and SCRIB facilitate S. Typhi uptake. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the gastroenteritis infection model. The impact of ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 function during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity. Author SummaryDuring infection, Salmonella manipulates host cells into engulfing the bacteria and establishing an intracellular niche. While many studies have identified genes involved in different stages of this Salmonella invasion process, few studies have examined how differences between human hosts contribute to infection susceptibility. Here we leveraged a candidate genetic screen to identify natural genetic variation in the human ARHGEF26 gene that correlates with Salmonella invasion. Springboarding from this result, we experimentally tested and revised existing models of ARHGEF26s role in Salmonella invasion, discovered an additional new role for ARHGEF26 during Salmonella disease, and confirmed our findings in mouse models. Building on how ARHGEF26 functions in other contexts, we implicated two ARHGEF26-interacting host proteins as contributors to Salmonella pathobiology. Collectively, these results identify a potential source of inter-person diversity in susceptibility to Salmonella disease, expand our molecular understanding of Salmonella infection to include a multifaceted role for ARHGEF26, and identify several important future directions that will be important to understand how Salmonella recruit and manipulate ARHGEF26 as well as how ARHGEF26 is able to drive Salmonella-beneficial processes.

microbiology↗