Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells

Holthenrich, Anna; Terglane, Julian; Naß, Johannes; Miętkowska, Magdalena; Kerkhoff, Eugen; Gerke, Volker

doi:10.1007/s00018-021-04108-x

Cited by 9 publications

(12 citation statements)

References 59 publications

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“…Extending these findings, our analysis of VWF secretion after Slp2-a depletion (Fig. S3) revealed a reduction of both, the histamine-triggered release of VWF to the cell culture medium and the amount of exocytosed VWF appearing on the cell surface, which was detected using an anti-VWF antibody capture assay [ 11 , 41 ] (Fig. 5 A).…”

Section: Resultsmentioning

confidence: 59%

Tip-end fusion of a rod-shaped secretory organelle

Naß

Koerdt

Biesemann

et al. 2022

Cell. Mol. Life Sci.

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Weibel–Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB–plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2.

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Section: Resultsmentioning

confidence: 59%

Tip-end fusion of a rod-shaped secretory organelle

Naß

Koerdt

Biesemann

et al. 2022

Cell. Mol. Life Sci.

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“…Actomyosin ring function occurs in discrete phases and is essential for unfurling platelet-catching VWF strings across the cell surface. 1 Ring recruitment is initiated by upstream signalling that promotes de novo nucleation 1,11 or rearrangement of actin filaments. 12 This is followed by contraction and finally disassembly.…”

Section: Main Textmentioning

confidence: 99%

“…Vc has been implicated in stabilising the actomyosin ring 11 and in other secretory systems additional myosins help recruit actin and organise contraction. 14,15 While the actomyosin ring is essential for controlling the haemostatic output of WPB, the copackaged cargo that supports an inflammatory response (e.g., P-selectin) is not affected.…”

Section: Main Textmentioning

confidence: 99%

Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor

El-Mansi

Robinson

Kostelnik

et al. 2023

Blood

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In response to tissue injury, within seconds the ultra large glycoprotein, von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel-Palade bodies) into the lumen of the blood vasculature where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring, that forms, contracts and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies though characterising proteins associated with such a dynamic process has been challenging. We have combined APEX-2 proximity labelling with an innovative dual loss of function screen to identify proteins associated with actomyosin ring function. We show p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events control actomyosin ring function aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

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“…Myo5c is widely distributed and most prominently produced in cells from intestinal and glandular tissues, including the colon, pancreas, mammary, thyroid, and different salivary glands. Myo5c is also present in various cell lines such as HeLa, HEK 293, Caco–2, or HUVEC, suggesting that Myo5c has a wide distribution among different cell types ( Rodriguez and Cheney, 2002 ; Holthenrich et al, 2022 ). In endothelial cells Myo5c is involved in the transport and membrane fusion of Weibel–Palade bodies containing von Willebrand factor, an important hemostatic factor ( Dolce et al, 2020 ; Holthenrich et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

“…Myo5c is also present in various cell lines such as HeLa, HEK 293, Caco–2, or HUVEC, suggesting that Myo5c has a wide distribution among different cell types ( Rodriguez and Cheney, 2002 ; Holthenrich et al, 2022 ). In endothelial cells Myo5c is involved in the transport and membrane fusion of Weibel–Palade bodies containing von Willebrand factor, an important hemostatic factor ( Dolce et al, 2020 ; Holthenrich et al, 2022 ). Multi–ciliated cells possess a large number of motile cilia that beat in a coordinated and polarized manner.…”

Section: Introductionmentioning

confidence: 99%

Motor properties of Myosin 5c are modulated by tropomyosin isoforms and inhibited by pentabromopseudilin

Kengyel,

Palarz,

Krohn

et al. 2024

Front. Physiol.

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Myosin 5c (Myo5c) is a motor protein that is produced in epithelial and glandular tissues, where it plays an important role in secretory processes. Myo5c is composed of two heavy chains, each containing a generic motor domain, an elongated neck domain consisting of a single α–helix with six IQ motifs, each of which binds to a calmodulin (CaM) or a myosin light chain from the EF–hand protein family, a coiled–coil dimer–forming region and a carboxyl–terminal globular tail domain. Although Myo5c is a low duty cycle motor, when two or more Myo5c–heavy meromyosin (HMM) molecules are linked together, they move processively along actin filaments. We describe the purification and functional characterization of human Myo5c–HMM co–produced either with CaM alone or with CaM and the essential and regulatory light chains Myl6 and Myl12b. We describe the extent to which cofilaments of actin and Tpm1.6, Tpm1.8 or Tpm3.1 alter the maximum actin–activated ATPase and motile activity of the recombinant Myo5c constructs. The small allosteric effector pentabromopseudilin (PBP), which is predicted to bind in a groove close to the actin and nucleotide binding site with a calculated ΔG of −18.44 kcal/mol, inhibits the motor function of Myo5c with a half–maximal concentration of 280 nM. Using immunohistochemical staining, we determined the distribution and exact localization of Myo5c in endothelial and endocrine cells from rat and human tissue. Particular high levels of Myo5c were observed in insulin–producing β–cells located within the pancreatic islets of Langerhans.

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Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells

Cited by 9 publications

References 59 publications

Tip-end fusion of a rod-shaped secretory organelle

Tip-end fusion of a rod-shaped secretory organelle

Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor

Motor properties of Myosin 5c are modulated by tropomyosin isoforms and inhibited by pentabromopseudilin

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