Rab1A promotes cell proliferation and migration by upregulating Gli1 in colorectal cancer

Rab1A, as a highly conserved small guanosine triphosphatase (GTPase), plays contentious roles in different types of cancers. The role of Rab1A in colorectal cancer (CRC) has been described in previous studies, but the molecular mechanisms of Rab1A in CRC remain far from being addressed. In the present study, we found that Rab1A expression was significantly upregulated in CRC tissues and increased Rab1A expression correlated with tumor size, lymph node metastasis (LNM) and tumor-node-metastasis (TNM) stage of CRC patients. We also found that Rab1A exerts its promotive effect on CRC cell proliferation, migration and EMT progress. Further mechanistic experiments showed that glioma-associated oncogene-1 (Gli1), as a key transcriptional factor of the Hedgehog pathway, was implicated in Rab1A-mediated regulation of CRC cell proliferation and migration. In addition, Rab1A upregulated Gli1 expression through Smoothened homolog (SMO)-independent pathway. Finally, Rab1A activated mechanistic target of rapamycin (mTOR) signaling in CRC cells. Collectively, our results define Rab1A as a novel regulator of Gli1 to promote CRC cell proliferation and migration, and suggest that the Rab1A/mTOR/Gli1 axis may serve as a promising therapeutic target for the treatment of CRC.

Astrocytes close the mouse critical period for visual plasticity

Brain postnatal development is characterized by critical periods of experience-dependent remodeling of neuronal circuits. Failure to end these periods results in neurodevelopmental disorders. The cellular processes defining critical-period timing remain unclear. Here, we show that in the mouse visual cortex, astrocytes control critical-period closure. We uncover the underlying pathway, which involves astrocytic regulation of the extracellular matrix, allowing interneuron maturation. Unconventional astrocyte connexin signaling hinders expression of extracellular matrix–degrading enzyme matrix metalloproteinase 9 (MMP9) through RhoA–guanosine triphosphatase activation. Thus, astrocytes not only influence the activity of single synapses but also are key elements in the experience-dependent wiring of brain circuits.

The lysosomal Rag-Ragulator complex licenses RIPK1– and caspase-8–mediated pyroptosis by Yersinia

Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor–β–activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide CRISPR screen to uncover mediators of caspase-8–dependent pyroptosis identified an unexpected role of the lysosomal folliculin (FLCN)–folliculin-interacting protein 2 (FNIP2)–Rag-Ragulator supercomplex, which regulates metabolic signaling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, Fas-associated death domain (FADD), RIPK1, and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag guanosine triphosphatase activity and lysosomal tethering of Rag-Ragulator but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.

Activation of Rac1-Mineralocorticoid Receptor Pathway Contributes to Renal Injury in Salt-Loaded db/db Mice

The progression of diabetic kidney disease (DKD), a leading cause of end-stage kidney disease, involves mineralocorticoid receptor (MR) activation. We previously identified crosstalk between the small guanosine triphosphatase (GTPase) RAS-related C3 botulinus toxin substrate 1 (Rac1) and MR, but the role of Rac1-MR pathway activation in the progression of DKD is not clear. We performed uninephrectomy on type 2 diabetic mouse models, db/db (UNx-high salt [HS] db/db ), and their lean control, db/m (UNx-HS db/m ), at 4-week postpartum, and fed them a high-salt diet for 10 weeks. To evaluate the involvement of the Rac1-MR pathway in the DKD progression, we investigated the effects of the nonsteroidal MR antagonist, finerenone, and the Rac1 inhibitor, NSC23766, on blood pressure and glomerular injury in UNx-HS db/db mice. UNx-HS db/db mice with hyperaldosteronism showed hypertension and hypokalemia with increased cleaved α-epithelial sodium channel expressions and massive albuminuria, accompanied by glomerular injury with nodular lesions, which is a characteristic finding in human diabetic nephropathy. Expressions of active Rac1 and serum-and glucocorticoid-induced protein kinase 1 (Sgk1), a downstream molecule of MR signaling, in the renal cortex and isolated glomeruli, significantly elevated in UNx-HS db/db mice, associated with intense staining of active Rac1 in glomerular podocytes, but both hypertension and renal injury were ameliorated by NSC23766 and finerenone, associated with Sgk1 inhibition, suggesting that Rac1-MR activation contributes to hypertension and podocyte injury. In conclusion, salt-induced activation of Rac1-MR pathway in distal tubules and glomeruli is involved in DKD progression through hypertension and glomerular injury, respectively. This finding highlights MR antagonism along with Rac1 inhibition as a novel strategy for DKD treatment.

Receptor compaction and GTPase rearrangement drive SRP-mediated cotranslational protein translocation into the ER

The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)–driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases.

Dynamin controls neuropeptide secretion by organizing dense-core vesicle fusion sites

Synaptic vesicles (SVs) release neurotransmitters at specialized active zones, but release sites and organizing principles for the other major secretory pathway, neuropeptide/neuromodulator release from dense-core vesicles (DCVs), remain elusive. We identify dynamins, yeast Vps1 orthologs, as DCV fusion site organizers in mammalian neurons. Genetic or pharmacological inactivation of all three dynamins strongly impaired DCV exocytosis, while SV exocytosis remained unaffected. Wild-type dynamin restored normal exocytosis but not guanosine triphosphatase–deficient or membrane-binding mutants that cause neurodevelopmental syndromes. During prolonged stimulation, repeated use of the same DCV fusion location was impaired in dynamin 1-3 triple knockout neurons. The syntaxin-1 staining efficiency, but not its expression level, was reduced. αSNAP (α–soluble N-ethylmaleimide–sensitive factor attachment protein) expression restored this. We conclude that mammalian dynamins organize DCV fusion sites, downstream of αSNAP, by regulating the equilibrium between fusogenic and non-fusogenic syntaxin-1 promoting its availability for SNARE (SNAP receptor) complex formation and DCV exocytosis.

CLIC1 and CLIC4 mediate endothelial S1P receptor signaling to facilitate Rac1 and RhoA activity and function

Chloride intracellular channels 1 (CLIC1) and 4 (CLIC4) are expressed in endothelial cells and regulate angiogenic behaviors in vitro, and the expression of Clic4 is important for vascular development and function in mice. Here, we found that CLIC1 and CLIC4 in endothelial cells regulate critical G protein–coupled receptor (GPCR) pathways associated with vascular development and disease. In cultured endothelial cells, we found that CLIC1 and CLIC4 transiently translocated to the plasma membrane in response to sphingosine 1-phosphate (S1P). Both CLIC1 and CLIC4 were essential for mediating S1P-induced activation of the small guanosine triphosphatase (GTPase) Rac1 downstream of S1P receptor 1 (S1PR1). In contrast, only CLIC1 was essential for S1P-induced activation of the small GTPase RhoA downstream of S1PR2 and S1PR3. Neither were required for other S1P-S1PR signaling outputs. Rescue experiments revealed that CLIC1 and CLIC4 were not functionally interchangeable, suggesting distinct and specific functions for CLICs in transducing GPCR signaling. These CLIC-mediated mechanisms were critical for S1P-induced stimulation of the barrier function in endothelial cell monolayers. Our results define CLICs as previously unknown players in the pathways linking GPCRs to small GTPases and vascular endothelial function.

Crystal Structure of the Epo1-Bem3 Complex for Bud Growth

Tubules of the endoplasmic reticulum (ER) spread into the buds of yeast by an actin-based mechanism and, upon entry, become attached to the polarisome, a proteinaceous micro-compartment below the tip of the bud. The minimal tether between polarisome and cortical ER is formed by a protein complex consisting of Epo1, a member of the polarisome, Scs2, a membrane protein of the ER and Cdc42 guanosine triphosphatase-activating protein Bem3. Here, we report the crystal structure of a complex between Epo1 and Bem3. In addition, we characterize through the hydrogen/deuterium (H/D) exchange assay the interface between Scs2 and Epo1. Our findings provide a first structural insight into the molecular architecture of the link between cortical ER and the polarisome.

Role of Rho in Salt-Sensitive Hypertension

A high amount of salt in the diet increases blood pressure (BP) and leads to salt-sensitive hypertension in individuals with impaired renal sodium excretion. Small guanosine triphosphatase (GTP)ase Rho and Rac, activated by salt intake, play important roles in the pathogenesis of salt-sensitive hypertension as key switches of intracellular signaling. Focusing on Rho, high salt intake in the central nervous system increases sodium concentrations of cerebrospinal fluid in salt-sensitive subjects via Rho/Rho kinase and renin-angiotensin system activation and causes increased brain salt sensitivity and sympathetic nerve outflow in BP control centers. In vascular smooth muscle cells, Rho-guanine nucleotide exchange factors and Rho determine sensitivity to vasoconstrictors such as angiotensin II (Ang II), and facilitate vasoconstriction via G-protein and Wnt pathways, leading to increased vascular resistance, including in the renal arteries, in salt-sensitive subjects with high salt intake. In the vascular endothelium, Rho/Rho kinase inhibits nitric oxide (NO) production and function, and high salt amounts further augment Rho activity via asymmetric dimethylarginine, an endogenous inhibitor of NO synthetase, causing aberrant relaxation and increased vascular tone. Rho-associated mechanisms are deeply involved in the development of salt-sensitive hypertension, and their further elucidation can help in developing effective protection and new therapies.

Mitochondrial Dynamics: Molecular Mechanisms, Related Primary Mitochondrial Disorders and Therapeutic Approaches

Mitochondria do not exist as individual entities in the cell—conversely, they constitute an interconnected community governed by the constant and opposite process of fission and fusion. The mitochondrial fission leads to the formation of smaller mitochondria, promoting the biogenesis of new organelles. On the other hand, following the fusion process, mitochondria appear as longer and interconnected tubules, which enhance the communication with other organelles. Both fission and fusion are carried out by a small number of highly conserved guanosine triphosphatase proteins and their interactors. Disruption of this equilibrium has been associated with several pathological conditions, ranging from cancer to neurodegeneration, and mutations in genes involved in mitochondrial fission and fusion have been reported to be the cause of a subset of neurogenetic disorders.