Microtubule‐Dependent Transport of Secretory Vesicles in RBL‐2H3 Cells

Smith, Alexander J.; Pfeiffer, Janet R.; Zhang, Jun; Martinez, A. M.; Griffiths, Gillian M.; Wilson, Bridget S.

doi:10.1034/j.1600-0854.2003.00084.x

Cited by 98 publications

(108 citation statements)

References 30 publications

(32 reference statements)

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“…Microtubules are involved in mast cell degranulation, because the movement of secretory granules depends on intact microtubules (16,17). This finding is supported by demonstrations that agents inhibiting tubulin polymerization also suppress degranulation (18)(19)(20).…”

supporting

confidence: 70%

“…Accumulating recent data point to an important role of microtubules in these processes (38). Previous studies focused primarily on the role of microtubules in granular transport (13,16,17,20) or on the initial stages of SOCE signaling pathway (13,15,39). In this study, we show that microtubule network rearrangement in activated BMMCs and formation of microtubule protrusions is dependent on the activity of Ca 2+ sensor STIM1.…”

Section: Discussionmentioning

confidence: 53%

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STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

Hájková

Bugajev

Dráberová

et al. 2011

The Journal of Immunology

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Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca2+ controlled by store-operated Ca2+ entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca2+. Changes in cytosolic Ca2+concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca2+ response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca2+ plays an important role in the formation of microtubule protrusions in BMMCs.

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supporting

confidence: 70%

Section: Discussionmentioning

confidence: 53%

STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

Hájková

Bugajev

Dráberová

et al. 2011

The Journal of Immunology

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“…Microtubules, built up from ab-tubulin heterodimers, are important for mast cell degranulation, because the movement of secretory granules depends on intact microtubules (2,3), and compounds inhibiting tubulin polymerization suppress degranulation (4). It was reported that FcεRI aggregation leads to reorganization of microtubules (3,5) and that the influx of Ca 2+ plays a decisive role in microtubule remodeling (6,7).…”

mentioning

confidence: 99%

Microtubule Nucleation in Mouse Bone Marrow–Derived Mast Cells Is Regulated by the Concerted Action of GIT1/βPIX Proteins and Calcium

Sulimenko

Hájková

Černohorská

et al. 2015

The Journal of Immunology

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Ag-mediated activation of mast cells initiates signaling events leading to Ca2+ response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow–derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor β (βPIX) and G protein–coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of βPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that βPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca2+ affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca2+ level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and βPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/βPIX proteins, and Ca2+ in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.

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“…Following antigen stimulation and receptor crosslinking the intracellular signaling cascade is initiated. This cascade results in secretory vesicles fuse to the cell membrane and release their contents into the extracellular space [2]. While the identity of these and other molecular players are known, as well as their relative roles in immune response signaling cascades, relatively little work has been done in studying immune response in the stochastic regime.…”

mentioning

confidence: 99%

“…Vital to a comprehensive understanding of immune responses is the early spatial-temporal behavior of proteins at the membrane. We chose to study the dynamics of the high-affinity IgE receptor (FcεRI) in mast cells [1,2] following addition of its stimulatory ligand dinitrophenol (DNP). This system represents a well-studied, yet not fully understood molecular mediator of acquired immune system stimulation.…”

mentioning

confidence: 99%

Imaging Adaptive Immune Response in Single Cells using TIRF Microscopy

Aaron

Carroll‐Portillo²,

Pfeiffer³

et al. 2009

Microsc Microanal

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The complex trafficking of membrane-associated proteins plays an important role in antigen recognition and immune response. Vital to a comprehensive understanding of immune responses is the early spatial-temporal behavior of proteins at the membrane. We chose to study the dynamics of the high-affinity IgE receptor (FcεRI) in mast cells [1,2] following addition of its stimulatory ligand dinitrophenol (DNP). This system represents a well-studied, yet not fully understood molecular mediator of acquired immune system stimulation. Receptor dynamics and secretory vesicle trafficking were followed simultaneously in living cells with total internal reflection fluorescence microscopy (TIRF-M). We chose to visualize secretory lysosomes using the apoptosis-causing Fas ligand (FasL) as it has been shown to be targeted to secretory lysosomes within mast cells. Following antigen stimulation and receptor crosslinking the intracellular signaling cascade is initiated. This cascade results in secretory vesicles fuse to the cell membrane and release their contents into the extracellular space [2]. While the identity of these and other molecular players are known, as well as their relative roles in immune response signaling cascades, relatively little work has been done in studying immune response in the stochastic regime. Optical microscopy provides an opportunity to evaluate single cell behavior, thus providing a more complete, nuanced view of this system. TIRF microscopy, in particular, lends itself well to studies of membrane-associated proteins, as it confines the excitation to the cell-antigen interface [3]. We synthesized supported lipid bilayers containing up to 25l% (mol/mol) dinitrophenol (DNP). RBL mast cells previously transfected with FasL:GFP fusion protein, and incubated with Dy-IgE fluorescent conjugates were deposited on the DNP-lipid bilayers. Due to the large Stokes' shift of the Dy dye, both fluorophors were excited by a single 488nm laser and monitored simultaneously on a single CCD detector.Figures 1 and 2 highlight our results. Figure 1A contains TIRF images of IgE receptor clusters (shown in red) and FasL expression within the secretory vesicles (green). A large-scale reorganization towards the central portion of the interface is observed within 5-10 minutes of cell contact with the stimulatory substrate. We hypothesize that this is made possible by the 2D fluidity of the DNP-containing lipid bilayer. Similar large-scale IgE reorganization is not observed when antigen mobility is restricted via glutaraldehyde tethering of DNP to a glass surface (B, left and middle), or if stimulated cells are in contact with an untreated glass surface (B, right). Figure 2, left indicates DNP-mediated FasL degranulation (shown in green) in RBL cells at early time-points upon contact with the DNP-lipid bilayer. Images were acquired at 10fps. Upon fusing with the cell membrane, FasL-containing secretory vesicles become apparent as "bursts" in the signal intensity, as shown in Figure 2, right. Further image analysis will be pres...

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Microtubule‐Dependent Transport of Secretory Vesicles in RBL‐2H3 Cells

Cited by 98 publications

References 30 publications

STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

Microtubule Nucleation in Mouse Bone Marrow–Derived Mast Cells Is Regulated by the Concerted Action of GIT1/βPIX Proteins and Calcium

Imaging Adaptive Immune Response in Single Cells using TIRF Microscopy

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