Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented.
View Article and Find Full Text PDFThe nuclear pore complex (NPC) is the major conduit for nucleocytoplasmic transport and serves as a platform for gene regulation and DNA repair. Several nucleoporins undergo ubiquitylation and SUMOylation, and these modifications play an important role in nuclear pore dynamics and plasticity. Here, we perform a detailed analysis of these post-translational modifications of yeast nuclear basket proteins under normal growth conditions as well as upon cellular stresses, with a focus on SUMOylation.
View Article and Find Full Text PDF, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-β-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding.
View Article and Find Full Text PDFDuring mesenchymal cell motility, various actin regulators are recruited to the leading edge with exquisite precision in time and space to generate protrusion and retraction cycles. We present here an automated method, named CorRecD (from Correlation Recruitment Dynamics), which quantifies cell edge dynamics, protein recruitment and analyze their cross-correlation. The Wave Regulatory Complex (WRC), a master driver of protrusions, is used as a case-of-study.
View Article and Find Full Text PDFBackground: Characterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins.
View Article and Find Full Text PDFCoordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility.
View Article and Find Full Text PDFJ Opt Soc Am A Opt Image Sci Vis
October 2015
Fluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurements yield numerous indications on cellular processes such as interprotein and intraprotein mechanisms through fluorescent tagging and Förster resonance energy transfer. In this area, frequency-domain fluorescence lifetime imaging microscopy is particularly appropriate to probe a sample noninvasively and quantify these interactions in living cells.
View Article and Find Full Text PDFDissemination of carcinoma cells requires the pericellular degradation of the extracellular matrix, which is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP). In this article, we report a co-up-regulation and colocalization of MT1-MMP and atypical protein kinase C iota (aPKCι) in hormone receptor-negative breast tumors in association with a higher risk of metastasis. Silencing of aPKC in invasive breast-tumor cell lines impaired the delivery of MT1-MMP from late endocytic storage compartments to the surface and inhibited matrix degradation and invasion.
View Article and Find Full Text PDFEarly endosomes consist of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. The tubular transport intermediates that constitute recycling endosomes function in cell polarity, migration, and cytokinesis. Endosomal tubulation and fission require both actin and intact microtubules, but although factors that stabilize recycling endosomal tubules have been identified, those required for tubule generation from vacuolar sorting endosomes (SEs) remain unknown.
View Article and Find Full Text PDFParticle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios.
View Article and Find Full Text PDFLoss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants.
View Article and Find Full Text PDFIn eukaryotes, mRNA export involves many evolutionarily conserved factors that carry the nascent transcript to the nuclear pore complex (NPC). The THO/TREX complex couples transcription to mRNA export and recruits the mRNA export receptor NXF1 for the transport of messenger ribonucleoprotein particles (mRNP) to the NPC. The transcription and export complex 2 (TREX-2) was suggested to interact with NXF1 and to shuttle between transcription sites and the NPC.
View Article and Find Full Text PDFRegulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes.
View Article and Find Full Text PDFThe CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein.
View Article and Find Full Text PDFA large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2.
View Article and Find Full Text PDFIn natural, industrial and medical environments, microorganisms mainly live as structured and organised matrix-encased communities known as biofilms. In these communities, microorganisms demonstrate coordinated behaviour and are able to perform specific functions such as dramatic resistance to antimicrobials, which potentially lead to major public health and industrial problems. It is now recognised that the appearance of such specific biofilm functions is intimately related to the three-dimensional organisation of the biological edifice, and results from multifactorial processes.
View Article and Find Full Text PDFThe presence of multiple membrane-bound intracellular compartments is a major feature of eukaryotic cells. Many of the proteins required for formation and maintenance of these compartments share an evolutionary history. Here, we identify the SEA (Seh1-associated) protein complex in yeast that contains the nucleoporin Seh1 and Sec13, the latter subunit of both the nuclear pore complex and the COPII coating complex.
View Article and Find Full Text PDFInvasion across tissue boundaries by metastatic tumor cells depends on the proteolytic degradation of the extracellular matrix, initiated by the formation of invadopodia, actin-driven membrane protrusions with matrix-degradative activity. Yet, mechanisms underlying invadopodia formation remain largely unknown. In this report, we examined the role of the histone deacetylase HDAC6 in invadopodia formation and invasion by breast cancer cells.
View Article and Find Full Text PDFResearch about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS).
View Article and Find Full Text PDFRab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co-ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6-interacting protein 1 (R6IP1), originally identified as a Rab6-binding protein.
View Article and Find Full Text PDFStudies of proteins' interaction in cells by FRET can take benefit from two important photo-physical properties describing fluorescent proteins: fluorescence emission spectrum and fluorescence lifetime. These properties provide specific and complementary information about the tagged proteins and their environment. However, none of them taken individually can completely quantify the involved fluorophore characteristics due to their multiparametric dependency with molecular environment, experimental conditions, and interpretation complexity.
View Article and Find Full Text PDFBackground: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in Förster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed.
View Article and Find Full Text PDFThe molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1 microm3.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
October 2002
Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-membrane linker undergoes a series of conformational changes that modulate its interactions with various partners and its localization in membrane or cytosolic pools. Its mobility and exchange rates within and between these two pools were assessed by two-photon fluorescence recovery after photobleaching in epithelial cell microvilli.
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