scholarly journals Microvillar cartography: a super-resolution single-molecule imaging method to map the positions of membrane proteins with respect to cellular surface topography

2020 ◽  
Author(s):  
Shirsendu Ghosh ◽  
Ronen Alon ◽  
Andres Alcover ◽  
Gilad Haran
Keyword(s):  
Cell Surface ◽  
Single Molecule ◽  
Cell Activation ◽  
Super Resolution ◽  
Cell Receptor ◽  
Specific Protein ◽  
Surface Proteins ◽  
Imaging Method ◽  
Protein Distribution ◽  
Membrane Topography

AbstractWe introduce Microvillar Cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, carbon coating and immunogold clustering, and is amenable to future developments involving e.g. live-cell imaging. This Protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

scholarly journals Amyloid fibrils activate B-1a lymphocytes to ameliorate inflammatory brain disease

2015 ◽  
Vol 112 (49) ◽  
pp. 15016-15023 ◽  
Author(s):  
Michael Phillip Kurnellas ◽  
Eliver Eid Bou Ghosn ◽  
Jill M. Schartner ◽  
Jeanette Baker ◽  
Jesse J. Rothbard ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lymph Nodes ◽  
B Cell ◽  
T Cell Activation ◽  
Immune Suppression ◽  
Cell Activation ◽  
Amyloid Fibrils ◽  
Cell Receptor ◽  
Surface Proteins ◽  
Loss Of Function

Amyloid fibrils composed of peptides as short as six amino acids are therapeutic in experimental autoimmune encephalomyelitis (EAE), reducing paralysis and inflammation, while inducing several pathways of immune suppression. Intraperitoneal injection of fibrils selectively activates B-1a lymphocytes and two populations of resident macrophages (MΦs), increasing IL-10 production, and triggering their exodus from the peritoneum. The importance of IL-10–producing B-1a cells in this effective therapy was established in loss-of-function experiments where neither B-cell–deficient (μMT) nor IL10−/− mice with EAE responded to the fibrils. In gain-of-function experiments, B-1a cells, adoptively transferred to μMT mice with EAE, restored their therapeutic efficacy when Amylin 28–33 was administered. Stimulation of adoptively transferred bioluminescent MΦs and B-1a cells by amyloid fibrils resulted in rapid (within 60 min of injection) trafficking of both cell types to draining lymph nodes. Analysis of gene expression indicated that the fibrils activated the CD40/B-cell receptor pathway in B-1a cells and induced a set of immune-suppressive cell-surface proteins, including BTLA, IRF4, and Siglec G. Collectively, these data indicate that the fibrils activate B-1a cells and F4/80+ MΦs, resulting in their migration to the lymph nodes, where IL-10 and cell-surface receptors associated with immune-suppression limit antigen presentation and T-cell activation. These mechanisms culminate in reduction of paralytic signs of EAE.

scholarly journals Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors.

1993 ◽  
Vol 177 (1) ◽  
pp. 219-223 ◽  
Author(s):  
S Wee ◽  
G L Schieven ◽  
J M Kirihara ◽  
T T Tsu ◽  
J A Ledbetter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Cell Receptor ◽  
Surface Proteins ◽  
Cell Surface Receptors ◽  
Surface Receptors

When T cells are activated via the T cell receptor (TCR) complex a number of cellular substrates, including some cell surface proteins, become phosphorylated on tyrosine (Tyr) residues. Phosphorylation of cytoplasmic Tyr renders these cell surface receptors competent to interact with proteins that link cell surface receptors to protein in the intracellular signaling pathways. Here we show that Tyr residues in the cytoplasmic domain of CD6 become phosphorylated upon T cell activation via the TCR complex. Tyr phosphorylation was observed when the T cells were activated by crosslinking CD3 or by cocrosslinking CD3 with CD2 or CD4, but not when the cells were stimulated by crosslinking CD2, CD4, or CD28 alone. Unlike other Tyr kinase substrates, such as the phospholipase C gamma 1-associated pp35/36 protein, whose level of Tyr phosphorylation is highest when T cells are activated by cocrosslinking CD3 with CD2, the levels of CD6 Tyr phosphorylation are highest when T cells were activated by cocrosslinking CD3 with CD4.

scholarly journals A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation

2021 ◽  
Author(s):  
Jia C Wang ◽  
Yang-In Yim ◽  
Xufeng Wu ◽  
Valentin Jaumouillé ◽  
Clare M Waterman ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Activation ◽  
Synapse Formation ◽  
Super Resolution ◽  
Cell Receptor ◽  
B Cell Activation ◽  
Protein Distribution ◽  
Immune Synapse ◽  
Resolution Imaging ◽  
Membrane Bound

B-cell activation and immune synapse (IS) formation with membrane-bound antigens are actin-dependent processes that scale positively with the strength of antigen-induced signals. Importantly, ligating the B-cell integrin, LFA-1, with ICAM-1 promotes IS formation when antigen is limiting. Whether the actin cytoskeleton plays a specific role in integrin-dependent IS formation is unknown. Here we show using super-resolution imaging of primary B cells that LFA-1: ICAM-1 interactions promote the formation of an actomyosin network that dominates the B-cell IS. This network is created by the formin mDia1, organized into concentric, contractile arcs by myosin 2A, and flows inward at the same rate as B-cell receptor (BCR): antigen clusters. Consistently, individual BCR microclusters are swept inward by individual actomyosin arcs. Under conditions where integrin is required for synapse formation, inhibiting myosin impairs synapse formation, as evidenced by reduced antigen centralization, diminished BCR signaling, and defective signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network plays a key role in the mechanism by which LFA-1 co-stimulation promotes B-cell activation and IS formation.

scholarly journals Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data

2016 ◽  
Vol 27 (22) ◽  
pp. 3627-3636 ◽  
Author(s):  
Sophie V. Pageon ◽  
Philip R. Nicovich ◽  
Mahdie Mollazade ◽  
Thibault Tabarin ◽  
Katharina Gaus
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Cell Receptor ◽  
Intact Cells ◽  
Analysis Strategy ◽  
Signaling Activity

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.

scholarly journals Shared paramyxoviral glycoprotein architecture is adapted for diverse attachment strategies

2010 ◽  
Vol 38 (5) ◽  
pp. 1349-1355 ◽  
Author(s):  
Thomas A. Bowden ◽  
Max Crispin ◽  
E. Yvonne Jones ◽  
David I. Stuart
Keyword(s):  
Cell Surface ◽  
Viral Entry ◽  
Cell Attachment ◽  
Structural Information ◽  
Cell Receptor ◽  
Specific Protein ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Entry Inhibitors ◽  
Animal Pathogens

Members within the paramyxovirus subfamily Paramyxovirinae constitute a large number of highly virulent human and animal pathogens. The glycoproteins present on these viruses are responsible for mediating host cell attachment and fusion and are key targets for the design of antiviral entry inhibitors. In the present review, we discuss recent structural studies which have led to a better understanding of the various mechanisms by which different paramyxoviruses use their attachment glycoproteins to hijack specific protein and glycan cell-surface receptors to facilitate viral entry. It is observed that the paramyxovirus attachment glycoprotein consists of a conserved overall structure which includes an N-terminal six-bladed β-propeller domain which is responsible for cell receptor binding. Crystal structures of this domain from different biomedically important paramyxoviruses, including measles, Nipah, Hendra, Newcastle disease and parainfluenza viruses, alone and in complex with their functional cell-surface receptors, demonstrate three contrasting mechanisms of receptor engagement that paramyxoviruses have evolved to confer discreet protein- and glycan-receptor specificity. This structural information highlights the adaptability of the paramyxovirus attachment glycoprotein surface and the potential for the emergence of new and potentially harmful viruses in human hosts.

scholarly journals Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells

Nanoscale ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 10023-10033 ◽  
Author(s):  
Jan Bergstrand ◽  
Lei Xu ◽  
Xinyan Miao ◽  
Nailin Li ◽  
Ozan Öktem ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Dictionary Learning ◽  
Super Resolution ◽  
Distribution Patterns ◽  
Specific Protein ◽  
Protein Distribution ◽  
Activated Platelets ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Super-resolution imaging of P-selectin in platelets together with dictionary learning allow specifically activated platelets to be identified in an automatic objective manner.

scholarly journals Dynamic Regulation of a GPCR-Tetraspanin-G Protein Complex on Intact Cells: Central Role of CD81 in Facilitating GPR56-Gαq/11Association

2004 ◽  
Vol 15 (5) ◽  
pp. 2375-2387 ◽  
Author(s):  
Kevin D. Little ◽  
Martin E. Hemler ◽  
Christopher S. Stipp
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Cell Activation ◽  
Surface Proteins ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Scaffolding Proteins ◽  
Intact Cells ◽  
Heterotrimeric G Protein ◽  
Vast Number

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein–coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Gαq, Gα11, and Gβ, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Gαq/11complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Gαq/11complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.

scholarly journals Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

2016 ◽  
Vol 113 (5) ◽  
pp. E558-E567 ◽  
Author(s):  
Jing Wang ◽  
Shan Tang ◽  
Zhengpeng Wan ◽  
Yiren Gao ◽  
Yiyun Cao ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Molecule ◽  
Cell Activation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Antigen Binding ◽  
B Cell Activation ◽  
Class I Mhc ◽  
Antigen System

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

scholarly journals Downregulation of the T-Cell Receptor Complex and Impairment of T-Cell Activation by Human Herpesvirus 6 U24 Protein

2007 ◽  
Vol 82 (2) ◽  
pp. 602-608 ◽  
Author(s):  
Brian M. Sullivan ◽  
Laurent Coscoy
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Human Herpesvirus ◽  
Cell Receptor ◽  
Immune Recognition ◽  
Human Herpesvirus 6 ◽  
Herpesvirus 6

ABSTRACT We have performed a screen aimed at identifying human herpesvirus 6 (HHV-6)-encoded proteins that modulate immune recognition. Here we show that the U24 protein encoded by HHV-6 variant A downregulates cell surface expression of the T-cell receptor (TCR)/CD3 complex, a complex essential to T-cell activation and the generation of an immune adaptive response. In the presence of U24, the TCR/CD3 complex is endocytosed but is not recycled back to the plasma membrane. Instead, it accumulates in early and late endosomes. Interestingly, whereas CD3 downregulation from the cell surface is normally associated with T-cell activation, U24 downregulates CD3 independently of T-cell activation. Moreover, we found that U24-expressing T cells are resistant to activation by antigen-presenting cells. HHV-6 has evolved a unique mechanism of inhibition of T-cell activation that may impair the establishment of an adaptive immune response. Furthermore, lymphocyte activation creates an environment favorable to the reactivation and replication of lymphotropic herpesviruses. Thus, by inhibiting T-cell activation, HHV-6 might limit its reactivation and thus minimize immune recognition.

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