scholarly journals Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation

2020 ◽  
Vol 219 (11) ◽  
Author(s):  
Ross T.A. Pedersen ◽  
Julian E. Hassinger ◽  
Paul Marchando ◽  
David G. Drubin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Transition Point ◽  
Quantitative Imaging ◽  
Imaging Data ◽  
Cortical Actin ◽  
Initiation Phase ◽  
Spatial Regulation ◽  
Dependent Site

During clathrin-mediated endocytosis (CME), over 50 different proteins assemble on the plasma membrane to reshape it into a cargo-laden vesicle. It has long been assumed that cargo triggers local CME site assembly in Saccharomyces cerevisiae based on the discovery that cortical actin patches, which cluster near exocytic sites, are CME sites. Quantitative imaging data reported here lead to a radically different view of which CME steps are regulated and which steps are deterministic. We quantitatively and spatially describe progression through the CME pathway and pinpoint a cargo-sensitive regulatory transition point that governs progression from the initiation phase of CME to the internalization phase. Thus, site maturation, rather than site initiation, accounts for the previously observed polarized distribution of actin patches in this organism. While previous studies suggested that cargo ensures its own internalization by regulating either CME initiation rates or frequency of abortive events, our data instead identify maturation through a checkpoint in the pathway as the cargo-sensitive step.

scholarly journals Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation

2019 ◽  
Author(s):  
Ross TA Pedersen ◽  
Julian E Hassinger ◽  
Paul Marchando ◽  
David G Drubin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transition Point ◽  
Quantitative Imaging ◽  
Imaging Data ◽  
Cortical Actin ◽  
Initiation Phase ◽  
Spatial Regulation ◽  
Polarized Cells ◽  
Endocytosis Pathway ◽  
Dependent Site

AbstractDuring clathrin-mediated endocytosis (CME), over 50 different proteins assemble on the plasma membrane to reshape it into a cargo-laden vesicle. It has long been assumed that cargo triggers local CME site assembly in Saccharomyces cerevisiae based on the discovery that cortical actin patches clustered near exocytic sites are CME sites. Quantitative imaging data reported here lead to a radically different view of which CME steps are regulated and which steps are deterministic. We quantitatively and spatially describe progression through the CME pathway and pinpoint a cargo-sensitive regulatory transition point that governs progression from the initiation phase of CME to the internalization phase. Thus, site maturation, rather than site initiation, accounts for the previously observed polarized distribution of actin patches in this organism. While previous studies suggested that cargo ensures its own internalization by regulating either CME initiation rates or frequency of abortive events, our data instead identify maturation through a checkpoint in the pathway as the cargo-sensitive step.SummaryPedersen, Hassinger, et al. investigate steps of the clathrin-mediated endocytosis pathway that are subject to regulation. They report position-dependent differences in endocytic site maturation rates in polarized cells and suggest that cargo controls endocytic internalization through tuning site maturation rather than site initiation.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Incorporating radiomics into clinical trials: expert consensus on considerations for data-driven compared to biologically driven quantitative biomarkers

2021 ◽  
Author(s):  
Laure Fournier ◽  
Lena Costaridou ◽  
Luc Bidaut ◽  
Nicolas Michoux ◽  
Frederic E. Lecouvet ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Quantitative Imaging ◽  
A Priori ◽  
External Validation ◽  
Data Driven ◽  
Imaging Biomarkers ◽  
Clinical Validation ◽  
Biological Processes ◽  
Imaging Data ◽  
Selection Of

Abstract Existing quantitative imaging biomarkers (QIBs) are associated with known biological tissue characteristics and follow a well-understood path of technical, biological and clinical validation before incorporation into clinical trials. In radiomics, novel data-driven processes extract numerous visually imperceptible statistical features from the imaging data with no a priori assumptions on their correlation with biological processes. The selection of relevant features (radiomic signature) and incorporation into clinical trials therefore requires additional considerations to ensure meaningful imaging endpoints. Also, the number of radiomic features tested means that power calculations would result in sample sizes impossible to achieve within clinical trials. This article examines how the process of standardising and validating data-driven imaging biomarkers differs from those based on biological associations. Radiomic signatures are best developed initially on datasets that represent diversity of acquisition protocols as well as diversity of disease and of normal findings, rather than within clinical trials with standardised and optimised protocols as this would risk the selection of radiomic features being linked to the imaging process rather than the pathology. Normalisation through discretisation and feature harmonisation are essential pre-processing steps. Biological correlation may be performed after the technical and clinical validity of a radiomic signature is established, but is not mandatory. Feature selection may be part of discovery within a radiomics-specific trial or represent exploratory endpoints within an established trial; a previously validated radiomic signature may even be used as a primary/secondary endpoint, particularly if associations are demonstrated with specific biological processes and pathways being targeted within clinical trials. Key Points • Data-driven processes like radiomics risk false discoveries due to high-dimensionality of the dataset compared to sample size, making adequate diversity of the data, cross-validation and external validation essential to mitigate the risks of spurious associations and overfitting. • Use of radiomic signatures within clinical trials requires multistep standardisation of image acquisition, image analysis and data mining processes. • Biological correlation may be established after clinical validation but is not mandatory.

scholarly journals High yield expression of the Neurospora crassa plasma membrane H(+)-ATPase in Saccharomyces cerevisiae

1994 ◽  
Vol 269 (26) ◽  
pp. 17705-17712
Author(s):  
S.K. Mahanty ◽  
U.S. Rao ◽  
R.A. Nicholas ◽  
G.A. Scarborough
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Neurospora Crassa ◽  
High Yield

scholarly journals Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

1994 ◽  
Vol 125 (2) ◽  
pp. 381-391 ◽  
Author(s):  
J Mulholland ◽  
D Preuss ◽  
A Moon ◽  
A Wong ◽  
D Drubin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Ultrastructural Level ◽  
Actin Binding ◽  
Cortical Actin ◽  
Actin Binding Proteins ◽  
Double Labeling ◽  
Wall Growth ◽  
Cortical Cytoskeleton

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

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