Protein mobilities and P-selectin storage in Weibel–Palade bodies

Kiskin, Nikolai I.; Hellen, Nicola; Babich, Victor; Hewlett, Lindsay; Knipe, Laura; Hannah, Matthew J.; Carter, Tom

doi:10.1242/jcs.073593

Cited by 26 publications

(36 citation statements)

References 60 publications

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“…P-selectin is immobilised in the membrane of mature WPBs [9]. Here we show that P-selectin is re-mobilised after hormone-evoked transient plasma membrane fusion (Fig.…”

Section: Discussionsupporting

confidence: 56%

“…In similar experiments with ssEGFP in all n = 13 experiments on NH 4 Cl-rounded WPBs, despite even higher resolution ( D crit range 0.34–4.39 µm 2 /s, median 1.42 µm 2 /s), we also obtained D > D crit . Assuming the mobility in post-fusion rounded WPBs is similar to that in NH 4 Cl-rounded WPBs, these data suggest that small soluble proteins become highly mobile, with the mobilities possibly exceeding the range reported for Eotaxin-3-EGFP/ssEGFP in the endoplasmic reticulum of HUVEC, 1–2 µm 2 /s [9].…”

Section: Resultsmentioning

confidence: 61%

“…VWF-EGFP, Pro-EGFP, Pro-mRFP, tPA-EGFP, P-selectin-EGFP, EGFP-Rab27A, ssEGFP, EGFP-CD63, EGFP-Rab35 and Eotaxin-3-EGFP constructs were made or obtained as previously described [9], [10], [18]–[21]. Alexa Fluor 647 Hydrazide (Alexa-647) and Vibrant DiI (vDiI) were purchased from Invitrogen.…”

Section: Methodsmentioning

confidence: 99%

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Differential Cargo Mobilisation within Weibel-Palade Bodies after Transient Fusion with the Plasma Membrane

et al. 2014

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Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis.

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“…P-selectin is immobilised in the membrane of mature WPBs [9]. Here we show that P-selectin is re-mobilised after hormone-evoked transient plasma membrane fusion (Fig.…”

Section: Discussionsupporting

confidence: 56%

Section: Resultsmentioning

confidence: 61%

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Differential Cargo Mobilisation within Weibel-Palade Bodies after Transient Fusion with the Plasma Membrane

et al. 2014

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“…E). Alternatively, the partial inhibition could reflect a steric hindrance of the extracellular domain of P‐selectin as it exits the fusion pore; P‐selectin mobility is limited in mature WPBs . Therefore, the delivery of larger integral membrane proteins can be influenced by inhibition of the actin ring, although not enough to inhibit function (Fig.…”

Section: Resultsmentioning

confidence: 99%

Tuning the endothelial response: differential release of exocytic cargos from Weibel‐Palade bodies

Nightingale

McCormack

Grimes

et al. 2018

Journal of Thrombosis and Haemostasis

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Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation.The molecules that contribute to these processes are co‐stored in secretory granules.How can the cells control release of granule content to allow differentiated responses?Selected agonists recruit an exocytosis‐linked actin ring to boost release of a subset of cargo. SummaryBackgroundEndothelial cells harbor specialized storage organelles, Weibel‐Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P‐selectin. During full fusion, secretion of this large hemostatic protein and smaller pro‐inflammatory proteins are thought to be inextricably linked.ObjectiveTo determine if secretagogue‐dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis.MethodsWe used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high‐throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins.ResultsInhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms.ConclusionsSecretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo‐content release and the treatment of patients with von Willebrand disease.

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“…In addition, although the majority of LROs share certain characteristics, many of them display completely different features as well. Maturation stage of the cells must also be considered, since the recruitment of Rab27a is a dynamic process that depends on the maturation and polarization stage of the cell [45,47]. In this sense, for instance, when von Willebrand factor (VWF) is heterologously expressed in some cultured cell lines, such as HEK-293, it causes the formation of structures similar to WPBs that can recruit endogenous Rab27a.…”

Section: Discussionmentioning

confidence: 99%

Role of the small GTPase Rab27a during Herpes simplex virus infection of oligodendrocytic cells

et al. 2012

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BackgroundThe morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection.ResultsHerein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells.ConclusionsAltogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.

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Protein mobilities and P-selectin storage in Weibel–Palade bodies

Cited by 26 publications

References 60 publications

Differential Cargo Mobilisation within Weibel-Palade Bodies after Transient Fusion with the Plasma Membrane

Differential Cargo Mobilisation within Weibel-Palade Bodies after Transient Fusion with the Plasma Membrane

Tuning the endothelial response: differential release of exocytic cargos from Weibel‐Palade bodies

Role of the small GTPase Rab27a during Herpes simplex virus infection of oligodendrocytic cells

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