Kidney fibrosis is a highly deleterious process and a final manifestation of chronic kidney disease. Alpha-(α)-synuclein (SNCA) is an actin-binding neuronal protein with various functions within the ...brain; however, its role in other tissues is unknown. Here, we describe the expression of SNCA in renal epithelial cells and demonstrate its decrease in renal tubules of murine and human fibrotic kidneys, as well as its downregulation in renal proximal tubular epithelial cells (RPTECs) after TGF-β1 treatment. shRNA-mediated knockdown of SNCA in RPTECs results in de novo expression of vimentin and α-SMA, while SNCA overexpression represses TGF-β1-induced mesenchymal markers. Conditional gene silencing of SNCA in RPTECs leads to an exacerbated tubulointerstitial fibrosis (TIF) in two unrelated in vivo fibrotic models, which is associated with an increased activation of MAPK-p38 and PI3K-Akt pathways. Our study provides an evidence that disruption of SNCA signaling in RPTECs contributes to the pathogenesis of renal TIF by facilitating partial epithelial-to-mesenchymal transition and extracellular matrix accumulation.
Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and ...metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis.
Budding yeast cells are assumed to trigger Start and enter the cell cycle only after they attain a critical size set by external conditions. However, arguing against deterministic models of cell size ...control, cell volume at Start displays great individual variability even under constant conditions. Here we show that cell size at Start is robustly set at a single-cell level by the volume growth rate in G1, which explains the observed variability. We find that this growth-rate-dependent sizer is intimately hardwired into the Start network and the Ydj1 chaperone is key for setting cell size as a function of the individual growth rate. Mathematical modelling and experimental data indicate that a growth-rate-dependent sizer is sufficient to ensure size homeostasis and, as a remarkable advantage over a rigid sizer mechanism, it reduces noise in G1 length and provides an immediate solution for size adaptation to external conditions at a population level.
Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its ...complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.
The analysis of tumor interstitial fluid (TIF) composition is a valuable procedure to identify antimetastatic targets, and different laboratories have set up techniques for TIF isolation and ...proteomic analyses. However, those methods had never been compared in samples from the same tumor and patient. In this work, we compared the two most used methods, elution and centrifugation, in pieces of the same biopsy samples of cutaneous squamous cell carcinoma (cSCC). First, we established that high G-force (10 000g) was required to obtain TIF from cSCC by centrifugation. Second, we compared the centrifugation method with the elution method in pieces of three different cSCC tumors. We found that the mean protein intensities based in the number of peptide spectrum matches was significantly higher in the centrifuged samples than in the eluted samples. Regarding the robustness of the methods, we observed higher overlapping between both methods (77–80%) than among samples (50%). These results suggest that there exists an elevated consistence of TIF composition independently of the method used. However, we observed a 3-fold increase of extracellular proteins in nonoverlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies.
The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in ...yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition,
nse1-RING
mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.
Nuclear Cyclin D1 (Ccnd1) is a main regulator of cell cycle progression and cell proliferation. Interestingly, Ccnd1 moves to the cytoplasm at the onset of differentiation in neuronal precursors. ...However, cytoplasmic functions and targets of Ccnd1 in post-mitotic neurons are unknown. Here we identify the α4 subunit of gamma-aminobutyric acid (GABA) type A receptors (GABA
A
Rs) as an interactor and target of Ccnd1–Cdk4. Ccnd1 binds to an intracellular loop in α4 and, together with Cdk4, phosphorylates the α4 subunit at threonine 423 and serine 431. These modifications upregulate α4 surface levels, increasing the response of α4-containing GABA
A
Rs, measured in whole-cell patch-clamp recordings. In agreement with this role of Ccnd1–Cdk4 in neuronal signalling, inhibition of Cdk4 or expression of the non-phosphorylatable α4 decreases synaptic and extra-synaptic currents in the hippocampus of newborn rats. Moreover, according to α4 functions in synaptic pruning,
CCND1
knockout mice display an altered pattern of dendritic spines that is rescued by the phosphomimetic α4. Overall, our findings molecularly link Ccnd1–Cdk4 to GABA
A
Rs activity in the central nervous system and highlight a novel role for this G
1
cyclin in neuronal signalling.
G1 cyclin Cln3 plays a key role in linking cell growth and proliferation in budding yeast. It is generally assumed that Cln3, which is present throughout G1, accumulates passively in the nucleus ...until a threshold is reached to trigger cell cycle entry. We show here that Cln3 is retained bound to the ER in early G1 cells. ER retention requires binding of Cln3 to the cyclin-dependent kinase Cdc28, a fraction of which also associates to the ER. Cln3 contains a chaperone-regulatory J
i domain that counteracts Ydj1, a J chaperone essential for ER release and nuclear accumulation of Cln3 in late G1. Finally, Ydj1 is limiting for release of Cln3 and timely entry into the cell cycle. As protein synthesis and ribosome assembly rates compromise chaperone availability, we hypothesize that Ydj1 transmits growth capacity information to the cell cycle for setting efficient size/ploidy ratios.
PDF
