Biology subjects

Ginsberg, M.

Publications and source records attributed to Ginsberg, M..

7 recordsLinked to original sources

bioRxiv2026

Regulated RIAM-Talin Engagement Controls Adhesion Stability and Mechanical Output

RIAM (Rap1-GTP-interacting adaptor molecule) links Rap1 to talin-1 to promote integrin activation and is normally enriched at nascent adhesions. How the duration of RIAM-talin engagement influences adhesion dynamics and force transmission remains unclear. Here, we used a RIAM chimera in which the native talin-binding site was replaced with the talin-binding motif of Kank2 to enforce sustained talin association and redistribute RIAM modules to mature adhesions. This talin-tethered RIAM chimera enhanced integrin activation, accelerated both adhesion assembly and disassembly, reduced adhesion lifetime, increased the fraction of nascent adhesions nucleated by RIAM, and elevated cellular traction forces. Notably, while integrin activation by the chimera was largely Rap1-independent, Rap1 binding remained necessary for optimal adhesion turnover kinetics and full traction force generation. These findings demonstrate that prolonged talin engagement of RIAM is sufficient to reprogram adhesion dynamics and reveal a separable role for Rap1 in coordinating force transmission downstream of integrin activation. Together, our results highlight the importance of regulating the residence and engagement mode of RIAM at talin for controlling adhesion plasticity and mechanotransduction.

cell biology↗

bioRxiv2025

Silencing KRIT1 Partially Reverses the Effects of Disturbed Flow onThe Endothelial Cell Transcriptome

BackgroundEndothelial cells respond to forces generated by laminar blood flow with changes in vasodilation, anticoagulant, fibrinolytic, or anti-inflammatory functions which preserve vessel patency. These responses to flow sheer stress are primarily mediated by the modulation of transcription factors Kruppel-like factors 2 and 4 (KLF2 and KLF4). Notably, disturbed flow patterns, which are found in vascular areas predisposed to atherosclerosis, significantly reduce the endothelial expression of KLF2 and KLF4, resulting in changes in the transcriptome that exacerbate inflammation and thrombosis. The endothelial CCM complex, comprising KRIT1, CCM2, and CCM3, suppresses the expression of KLF2 and KLF4. Loss of function of the CCM complex has recently been suggested to protect from coronary atherosclerosis in humans. We thus hypothesized that silencing of KRIT1, the central scaffold of the CCM complex, can normalize the atherogenic effects of disturbed flow on the human endothelial transcriptome MethodsBulk RNA sequencing (RNA-seq) was conducted on human umbilical vein endothelial cells (HUVECs) after the expression of KRIT1 was silenced using specific siRNAs. The endothelial cells were exposed to three different conditions for 24 hours: pulsatile shear stress (laminar flow), oscillatory shear stress (disturbed flow), and static conditions (no flow). ResultsWe found that silencing KRIT1 expression in HUVECs restored the expression of the transcription factors KLF2 and KLF4 under oscillatory shear stress. This treatment resulted in a transcriptomic profile similar to that of endothelial cells under pulsatile shear stress. These findings suggest that inhibition of the CCM complex in endothelium plays a vasoprotective role by reactivating a protective gene program to help endothelial cells resist disturbed blood flow. ConclusionsTargeting CCM genes can activate well-known vasoprotective gene programs that enhance endothelial resilience to inflammation, hypoxia, and angiogenesis under disturbed flow conditions, providing a novel pathway for preventing atherosclerosis. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/642862v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@a20119org.highwire.dtl.DTLVardef@1f62f68org.highwire.dtl.DTLVardef@1a26d71org.highwire.dtl.DTLVardef@bf3c82_HPS_FORMAT_FIGEXP M_FIG C_FIG Endothelial cells respond to blood flow by expressing transcription factors such as Kruppel-Like Factors 2 and 4 (KLF2 and KLF4). Under oscillatory shear stress (OS) conditions, characterized by disturbed flow, KLF2 and KLF4 are suppressed in endothelial cells, which is linked to an atheroprone gene program that includes increased inflammation and risk of atherosclerosis (unhealthy endothelium). Our work shows that silencing of endothelial KRIT1 under OS increases expression of KLF2 and KLF4 mRNA levels as well as the expression of a protective gene program associated with resilience to inflammation, hypoxia, and angiogenesis, similar to those observed under pulsatile shear stress conditions (healthy endothelium).

pathology↗

bioRxiv2021

Phostensin Enables Lymphocyte Integrin Activation and Population of Peripheral Lymphoid Organs

Rap1 GTPase drives assembly of the Mig-10/RIAM/lamellipodin-Integrin-Talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (PTSN), a regulatory subunit of protein phosphatase 1, is a component of the complex. PTSN mediates de-phosphorylation of Rap1 thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes PTSN, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable to defective activation of integrins L{beta}2 and 4{beta}7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, PTSN enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of PTSN ameliorates T cell-mediated colitis. SUMMARYPhostensin, a protein phosphatase 1 regulatory subunit, supports lymphocyte integrin-dependent functions by mediating dephosphorylation of Rap1 to stabilize the MIT complex thereby enabling the population of peripheral lymphoid organs and T cell-mediated colitis.

immunology↗

bioRxiv2021

Non cell-autonomous effect of astrocytes on cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we report that proliferative astrocytes play a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2 and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilizes HIF-1 in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We show that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1 target gene and a known component of the hypoxic program, contributes to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevents progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

neuroscience↗

bioRxiv2020

Abortive Intussusceptive Angiogenesis Causes Multi-Cavernous Vascular Malformations

Mosaic inactivation of CCM2 in humans causes cerebral cavernous malformations (CCMs) containing adjacent dilated blood-filled multi-cavernous lesions. We used CRISPR-Cas9 mutagenesis to induce mosaic inactivation of zebrafish ccm2 resulting in a novel lethal multi-cavernous lesion in the embryonic caudal venous plexus (CVP) caused by obstruction of blood flow by intraluminal pillars. These pillars mimic those that mediate intussusceptive angiogenesis; however, in contrast to the normal process, the pillars failed to fuse to split the pre-existing vessel in two. Abortive intussusceptive angiogenesis stemmed from mosaic inactivation of ccm2 leading to patchy klf2a over-expression and resultant aberrant flow signaling. Surviving adult fish manifested histologically-typical hemorrhagic CCM. Formation of mammalian CCM requires the flow-regulated transcription factor KLF2; fish CCM and the embryonic CVP lesion failed to form in klf2a null fish indicating a common pathogenesis with the mammalian lesion. These studies describe a zebrafish CCM model and establish a mechanism that can explain the formation of characteristic multi-cavernous lesions.

developmental biology↗

bioRxiv2019

Endothelial struts, a mechanism to generate large lumenized blood vessels de novo

Lumenization of de novo formed blood vessels occurs either through cell hollowing (intracellular lumen)1-3 or cord hollowing (extracellular lumen)4-6 and restricts thereby the initial lumen diameter to one or two endothelial cells (ECs) respectively. However, vasculogenesis can result in large diameter blood vessels, raising the question how these vessels are formed. Here, we describe an alternative model of vasculogenesis that results in the formation of large diameter vessels. In this model, ECs coalesce into a branched network of EC struts within the future lumen of the vessel. These struts maintain the patency of the vessel and serve as a scaffold for the ECs forming the vessel wall, which initially consists out of a few patches of ECs. Together, we show that endothelial struts facilitate the formation of large blood vessels without being bound by the prerequisite of a cord-like structure, nor are they restricted in size.

developmental biology↗

bioRxiv2018

A Rap1 binding site and lipid-dependent helix in talin F1 domain cooperate in integrin activation

Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a negligible contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain contains a previously undetected Rap1 binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that transforms into an amphipathic helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical as charge reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1 binding site and a lipid-dependent amphipathic helix in talin1 F1 cooperate to enable a direct Rap1-talin1 interaction to cause integrin activation.\n\nSummaryThis work reveals that Rap1 GTPases bind directly to talin1 F1 domain and by cooperating with a unique lipid-dependent amphipathic helix in the F1 domain effects talin1-mediated integrin activation.

cell biology↗