The munc13-4–rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane

Blood ◽  
2011 ◽  
Vol 118 (6) ◽  
pp. 1570-1578 ◽  
Author(s):  
Edo D. Elstak ◽  
Maaike Neeft ◽  
Nadine T. Nehme ◽  
Jarno Voortman ◽  
Marc Cheung ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immune Cells ◽  
Total Internal Reflection ◽  
Target Cells ◽  
Binding Motif ◽  
Microscopy Imaging ◽  
Secretory Lysosome ◽  
Mast Cell Line ◽  
Type 3 ◽  
Secretory Lysosomes

Abstract Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4–rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4–rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.

scholarly journals Munc13-4 Is an Effector of Rab27a and Controls Secretion of Lysosomes in Hematopoietic Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 731-741 ◽  
Author(s):  
Maaike Neeft ◽  
Marnix Wieffer ◽  
Arjan S. de Jong ◽  
Gabriela Negroiu ◽  
Corina H.G. Metz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Genetic Disorder ◽  
Hematopoietic Cells ◽  
Lytic Granules ◽  
Secretory Lysosome ◽  
Griscelli Syndrome ◽  
Life Threatening ◽  
Lytic Granule ◽  
Secretory Lysosomes

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Δ608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.

scholarly journals Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

2017 ◽  
Vol 28 (6) ◽  
pp. 792-808 ◽  
Author(s):  
Sang Su Woo ◽  
Declan J. James ◽  
Thomas F. J. Martin
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Direct Evidence ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Microscopy Imaging ◽  
C2 Domains ◽  
Protein Receptor ◽  
Compound Exocytosis ◽  
Sensitive Factor

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells.

scholarly journals Sialic Acids and Their Influence on Human NK Cell Function

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 263
Author(s):  
Philip Rosenstock ◽  
Thomas Kaufmann
Keyword(s):  
Nk Cells ◽  
Immune Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Sialic Acids ◽  
Innate Immune ◽  
Target Cells ◽  
Carbon Backbone ◽  
Nk Cell Receptors ◽  
Cell Inhibition

Sialic acids are sugars with a nine-carbon backbone, present on the surface of all cells in humans, including immune cells and their target cells, with various functions. Natural Killer (NK) cells are cells of the innate immune system, capable of killing virus-infected and tumor cells. Sialic acids can influence the interaction of NK cells with potential targets in several ways. Different NK cell receptors can bind sialic acids, leading to NK cell inhibition or activation. Moreover, NK cells have sialic acids on their surface, which can regulate receptor abundance and activity. This review is focused on how sialic acids on NK cells and their target cells are involved in NK cell function.

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

scholarly journals Sites and Determinants of Early Cleavages in the Proteolytic Processing Pathway of Reovirus Surface Protein σ3

2002 ◽  
Vol 76 (10) ◽  
pp. 5184-5197 ◽  
Author(s):  
Judit Jané-Valbuena ◽  
Laura A. Breun ◽  
Leslie A. Schiff ◽  
Max L. Nibert
Keyword(s):  
Structural Changes ◽  
Proper Time ◽  
Strain Differences ◽  
Surface Protein ◽  
Cellular Membrane ◽  
Proteolytic Processing ◽  
Target Cells ◽  
Inactive Form ◽  
Reovirus Type ◽  
Type 3

ABSTRACT Entry of mammalian reovirus virions into target cells requires proteolytic processing of surface protein σ3. In the virion, σ3 mostly covers the membrane-penetration protein μ1, appearing to keep it in an inactive form and to prevent it from interacting with the cellular membrane until the proper time in infection. The molecular mechanism by which σ3 maintains μ1 in this inactive state and the structural changes that accompany σ3 processing and μ1 activation, however, are not well understood. In this study we characterized the early steps in σ3 processing and determined their effects on μ1 function and particle infectivity. We identified two regions of high protease sensitivity, “hypersensitive” regions located at residues 208 to 214 and 238 to 244, within which all proteases tested selectively cleaved σ3 as an early step in processing. Further processing of σ3 was required for infection, consistent with the fact that the fragments resulting from these early cleavages remained bound to the particles. Reovirus type 1 Lang (T1L), type 3 Dearing (T3D), and T1L × T3D reassortant virions differed in the sites of early σ3 cleavage, with T1L σ3 being cleaved mainly at residues 238 to 244 and T3D σ3 being cleaved mainly at residues 208 to 214. These virions also differed in the rates at which the early cleavages occurred, with cleavage of T1L σ3 occurring faster than cleavage of T3D σ3. Analyses using chimeric and site-directed mutants of recombinant σ3 identified carboxy-proximal residues 344, 347, and 353 as the primary determinants of these strain differences. The spatial relationships between these more carboxy-proximal residues and the hypersensitive regions were discerned from the σ3 crystal structure. The results indicate that proteolytic processing of σ3 during reovirus disassembly is a multistep pathway with a number of molecular determinants.

scholarly journals Munc13-4*rab27 complex tethers secretory lysosomes at the plasma membrane

2012 ◽  
Vol 5 (1) ◽  
pp. 64-67 ◽  
Author(s):  
Edo D. Elstak ◽  
Maaike Neeft ◽  
Nadine T. Nehme ◽  
Isabelle Callebaut ◽  
Geneviève de Saint Basile ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Lysosomes

Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

2006 ◽  
Vol 291 (1) ◽  
pp. G146-G155 ◽  
Author(s):  
Jong Hak Won ◽  
David I. Yule
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Acinar Cells ◽  
Internal Reflection ◽  
Pancreatic Acinar Cells ◽  
Wide Field ◽  
Exocrine Cells ◽  
Parotid Acinar Cells ◽  
Signaling Dynamics ◽  
Total Internal Reflection Microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.

scholarly journals Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

2006 ◽  
Vol 128 (5) ◽  
pp. 509-522 ◽  
Author(s):  
Alexander T. Stein ◽  
Carmen A. Ufret-Vincenty ◽  
Li Hua ◽  
Luis F. Santana ◽  
Sharona E. Gordon
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Specific Inhibitor ◽  
Thermal Hyperalgesia ◽  
Hek293 Cells ◽  
Drg Neurons ◽  
Excised Patches ◽  
Phosphoinositide 3 Kinase ◽  
Inside Out

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85β subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85β coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

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