Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane

Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475–487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission

A mechanosensing mechanism controls plasma membrane shape homeostasis at the nanoscale

As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.Spanish Ministry of Science and Innovation (PGC2018-099645-B-I00 to XT, PID2019-110298GB-I00 to PR-C and BFU2016-79916-P to XQ). European Commission (H2020-FETPROACT-01-2016-731957) European Research Council (Adv-883739 to XT). Generalitat de Catalunya (2021 SGR 01425 to XT and PR-C). The prize 'ICREA Academia' for excellence in research to PR-Cand MA. Fundació la Marató de TV3 (201936-30-31). Obra Social 'La Caixa' (agreement LCF/PR/HR20/52400004). IBEC is recipient of a Severo Ochoa Award of Excellence from the MINCIN. AC was supported by a FPU fellowship from Ministerio de Educación, Cultura y Deporte (Spain). Grant BFU2015-66785-P from the Ministerio de Economía y Competitividad (Spain) to FT. Associazione Italiana per la Ricerca sul Cancro AIRC-IG 18621 and 5XMille22759 to GS. The Italian Ministry of University and Scientific Research (PRIN 2017-Prot. 2017HWTP2K to GS).Peer reviewe

Smoking cessation opportunities in severe mental illness (tobacco intensive motivational and estimate risk — TIMER—): study protocol for a randomized controlled trial

There is an increased risk of premature death in people with severe mental illness (SMI). Respiratory disorders and cardiovascular disease are leading causes of increased mortality rates in these patients, and tobacco consumption remains the most preventable risk factor involved. Developing new tools to motivate patients towards cessation of smoking is a high priority. Information on the motivational value of giving the lung age and prevention opportunities is unknown in this high-risk population. In the context of community care, screening and early detection of lung damage could potentially be used, together with mobile technology, in order to produce a prevention message, which may provide patients with SMI with a better chance of quitting smoking.This study receives funding by the Spanish Ministry of Economy, Industry and Competitiveness, Instituto Carlos III (FIS PI16/00802)

DNA methylation epigenotypes in breast cancer molecular subtypes

12 páginas, 3 figuras, 3 tablas.-- et al.[Introduction]: Identification of gene expression-based breast cancer subtypes is considered a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene-expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and only a limited understanding exists of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and to deliver specific epigenotypes associated with particular breast cancer subtypes. [Methods]: By using a microarray approach, we analyzed DNA methylation in regulatory regions of 806 cancer-related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biologic validation by pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A, and 48 luminal B paired breast cancer/adjacent tissues. With the all-subset selection method, we identified the most subtype-predictive methylation profiles in multivariable logistic regression analysis. [Results]: The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identified novel subtype-specific epigenotypes that clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. [Conclusions]: Our results provide evidence that well-defined DNA methylation profiles enable breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer.This work was supported by grants from project CGL2008-01131 (Departamento de Sanidad del Gobierno Vasco), S-PE08UN45 and PE09BF02 (Departamento de Ciencia y Tecnologia del Gobierno Vasco), BIO2008-04212, and RD06/0020/1019 (Red Tematica de Investigacion Cooperativa en Cancer, RTICC) from the MICINN. The CIBER de Enfermedades Raras is an initiative of the ISCIII. NGB had a doctoral fellowship from the Basque Government (Departamento de Educacion, Universidades e Investigacion).Peer reviewe

The role of the ER Sterol Exit Sites (ERSES) in the asymmetric control of sterol-dependent endocytosis

Trabajo presentado en el DFG International Meeting2021- Life in between: the cell biology of interfaces, celebrado en modalidad virtual y presencial en Münster (Alemania) del 27 al 29 de septiembre de 2021.Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)¿endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion

Zooming in on the molecular mechanisms of endocytic budding by time-resolved electron microscopy

Endocytic budding implies the remodeling of a plasma membrane portion from a flat sheet to a closed vesicle. Clathrin- and actin-mediated endocytosis in yeast has proven a very powerful model to study this process, with more than 60 evolutionarily conserved proteins involved in fashioning primary endocytic vesicles. Major progress in the field has been made during the last decades by defining the sequential recruitment of the endocytic machinery at the cell cortex using live-cell fluorescence microscopy. Higher spatial resolution has been recently achieved by developing time-resolved electron microscopy methods, allowing for the first time the visualization of changes in the plasma membrane shape, coupled to the dynamics of the endocytic machinery. Here, we highlight these advances and review recent findings from yeast and mammals that have increased our understanding of where and how endocytic proteins may apply force to remodel the plasma membrane during different stages of the process. © 2013 Springer BaselThis work was supported by BFU2011-30185 and CSD2009-00016 grants from the Spanish Government to M.I.G.Peer Reviewe

Function and regulation of Saccharomyces cerevisiae myosins-I in endocytic budding

Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-IMyo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites. ©The Authors Journal compilation ©2011 Biochemical Society.This work was supported by the Ministerio de Ciencia e Innovación [grant numbers BFU2008-03500 and CSD2009-00016 (to M.I.G.)].Peer Reviewe

Storage-protein hydrolysis and protein-body breakdown in germinatedZea mays L. seeds

Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein 1. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.This work was supported by grant from the Consejo Superior de Investigaciones Cientificas and the Centro para el Desarrollo Tecnologico Industrial. M. Isabel Geli is the recipient of a fellowship from CSIC.Peer reviewe

Two Structural Domains Mediate Two Sequential Events in [gamma]-Zein Targeting: Protein Endoplasmic Reticulum Retention and Protein Body Formation

12 pages, 7 figures.-- PMID: 12244234 [PubMed].-- PMCID: PMC160571.[gamma]-Zein is a maize storage protein synthesized by endosperm cells and stored together with [alpha]- and [beta]-zeins in specialized organelles called protein bodies. Previous studies have shown that in maize there is only one type of protein body and it is derived directly from the endoplasmic reticulum (ER). In this article, we describe the domains of [gamma]-zein involved in ER retention and the domains involved in protein body formation. To identify the signal responsible for [gamma]-zein retention in ER-derived protein bodies, DNAs encoding various deletion mutants of [gamma]-zein were constructed and introduced into Arabidopsis as a heterologous system. By using pulse-chase experiments and immunoelectron microscopy, we demonstrated that the deletion of a proline-rich domain at the N terminus of [gamma]-zein puts an end to its retention in the ER; this resulted in the secretion of the mutated protein. The amino acid sequence of [gamma]-zein necessary for ER retention is the repeat domain composed of eight units of the hexapeptide PPPVHL. In addition, we observed that only those [gamma]-zein mutants that contained both the proline-rich repeat domain and the C-terminal cysteine-rich domain were able to form ER-derived protein bodies. We suggest that the retention of [gamma]-zein in the ER could be a result of a protein-protein association or a transient interaction of the repeat domain with ER membranes.This work was supported by Grant No. Bio92/186 from the Comision Interministerial de Ciencia y Technología (CICYT). M.I.G. is the recipient of afellowship from the Formación de1 Personal Investigador (FPI).Peer reviewe

Ultrastructural dynamics of proteins involved in endocytic budding

Fluorescence live-cell imaging has temporally resolved the conserved choreography ofmore than 30 proteins involved in clathrin and actin-mediated endocytic budding from the plasma membrane. However, the resolution of these studies is insufficient to unveil how the endocytic machinery actually drives membrane deformation in vivo. In this study, we use quantitative immuno-EM to introduce the temporal dimension to the ultrastructural analysis ofmembrane budding and define changes in the topography of the lipid bilayer coupled to the dynamics of endocytic proteins with unprecedented spatiotemporal resolution. Using this approach, we frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and show that constriction of the invaginations correlates with translocation ofmembrane-sculpting proteins. Furthermore,we showthat initial bending of the plasma membrane is independent of actin and clathrin polymerization and precedes building of an actin cap branched by the Arp2/3 complex. Finally, our data indicate that constriction and additional elongation of the endocytic profiles require themechanochemical activity of themyosins-I. Altogether, this work provides major insights into the molecularmechanisms driving membrane deformation in a cellular context.This work was supported by Grants MTM2009-10893 (to A.E.), and BFU2008-03500, BFU2011-30185, and CSD2009-00016 (to M.I.G.) from the Spanish Government.Peer Reviewe