Biology subjects

Francis, C. R.

Publications and source records attributed to Francis, C. R..

5 recordsLinked to original sources

bioRxiv2023

Arf6 Regulates Endocytosis and Angiogenesis by Promoting Filamentous Actin Assembly

Clathrin-mediated endocytosis (CME) is a process vital to angiogenesis as well as general vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of CME have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for CME. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide a analysis of Arf6s function in angiogenic endothelium, focusing on its role in lumenogenesis as well as its relation to actin and CME. We found that Arf6 localized to both filamentous actin and sites of CME in 2-dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, and this may be the primary driver underlying gross dysmorphogenesis during angiogenic sprouting in its absence. Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and CME.

developmental biology↗

bioRxiv2022

Rab35 Governs Apicobasal Polarity Through Regulation of Actin Dynamics During Sprouting Angiogenesis

In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35s primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization, which is required to setup proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent regulator of actin architecture during blood vessel development.

developmental biology↗

bioRxiv2021

Capturing Membrane Trafficking Events During 3D Angiogenic Development in Vitro

ObjectivesMechanisms that govern angiogenesis are paramount to our understanding of how blood vessels are formed embryonically, maintained in adulthood and manifest disease. Akin to transcriptional regulation of endothelial-specific genes, vesicular trafficking events dictate protein localization, functional activity, and half-life, providing a critically important regulatory step. However, there is little information detailing endothelial-specific trafficking signatures. This is due, in part, by limitations in visualizing trafficking events in endothelial tissues. Our aim in this investigation was to explore the use of a 3-dimensional (3D) in vitro sprouting model to image and evaluate membrane trafficking events compared to the conventional 2-dimensional (2D)-based culture method. MethodsPrimary Human endothelial cells were challenged to make multicellular sprouts using a fibrin-bead assay. An assortment of cell polarity and Rab proteins were quantified via immunocytochemistry and live-imaging to compare their localization between 3D sprouts and 2D culture. ResultsOur results show that sprouts generated from the fibrin-bead assay grow close to the imaging plane allowing for an orthogonal view of apical and basal membrane domains. Compared with 2D culture in which the apical and basal domains are in the axial orientation, limiting resolution, 3D sprouts are acquired in the X-Y plane providing high-resolution for viewing trafficking events. Second, we demonstrate that fibrin-bead generated sprouts have a strong apicobasal polarity axis. Third, we directly compare imaging of trafficking mediators podocalyxin and Rab35 between 3D sprouts and 2D culture. Here, we show that 3D sprouting structures are well-suited to capture trafficking events that are not present in 2D culture due to the lack of a defined apical domain. Lastly, we compared exocytic events of von Willebrand Factor between 3D sprouting and 2D culture. Our results demonstrate a distinct imaging advantage for monitoring these trafficking programs in 3D sprouts as compared with conventional 2D culture. ConclusionsIn general, our results establish that the fibrin-bead sprouting assay is well-suited for sub-cellular imaging of trafficking events during angiogenic growth. Additionally, the 2D endothelial culture does not enforce the formation of an apicobasal polarity axis.

cell biology↗

bioRxiv2021

Synaptotagmin-like protein 2a regulates lumen formation via Weibel-Palade body apical secretion of angiopoietin-2 during angiogenesis

ObjectiveVascular lumen formation requires the redistribution of intracellular proteins to instruct apico-basal polarity, thereby enforcing maturation of both luminal and basal domains. In the absence of proper apical signaling, lumen formation can be distorted leading to lumen collapse and cessation of blood flow. Synaptotagmin-like protein-2a (Slp2a) has been implicated in apical membrane signaling; however, the role of Slp2a in vascular lumen formation has never been assessed. Approach and ResultsOur results demonstrate that Slp2a is required for vascular lumen formation. Using a 3- dimensional sprouting assay, sub-cellular imaging, and zebrafish blood vessel development we establish that Slp2a resides at the apical membrane acting as a tether for Rab27a that decorates Weibel-Palade bodies (WPBs). Unique to endothelial tissue, we show that Slp2a regulates exocytic activity of WPBs, thus regulating release of WPB contents into the luminal space during angiogenesis. Angiopoietin-2 is a Tie-2 receptor ligand that is selectively released from WPB secretory granules. We identify a critical role for angiopoietin-2 in regulating endothelial lumenization and show that in the absence of Slp2a, WPB contents cannot fuse with the apical membrane. This disrupts the release of angiopoietin-2 and blocks Tie-2 signaling necessary for proper lumen formation. ConclusionsOur results demonstrate a novel requirement of Slp2a for vascular lumen formation. Moreover, we show that Slp2a is required for the exocytic release of WPB secretory granule cargo during vascular lumen development, and thus is a core upstream component of the WPB secretory pathway. Furthermore, we provide evidence that WPB-housed angiopoietin-2 is required for vascular lumen formation. HIGHLIGHTSO_LISynaptotagmin-like protein-2a (Slp2a) is required for vascular lumen formation via its interaction with Rab27a and Weibel Palade Body secretory granules. C_LIO_LISlp2a is recruited to the apical membrane where it regulates secretion of Weibel Palade Body components into the luminal space. C_LIO_LIIn the absence of Slp2a, Weibel Palade Body-housed angiopoietin-2 ligand cannot be exocytosed, this impedes activation of Tie-2 signaling required for lumen biogenesis. C_LIO_LIKnockout of Slp2a or Tie-2 in zebrafish blunts the formation of vascular lumens during angiogenic development. C_LI

developmental biology↗

bioRxiv2020

EHBP1 and EHD2 regulate Dll4 caveolin-mediated endocytosis during blood vessel development

Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear. Here, we identify EHD2 and EHBP1 as novel regulators of Notch activation in endothelial cells through controlling endocytosis of Delta-like ligand 4 (Dll4). Knockout of EHBP1 and EHD2 in zebrafish produced a significant increase in ectopic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. In vitro, EHBP1 and EHD2 localized to plasma membrane-bound Dll4 and actin independently of clathrin. Disruption of caveolin endocytosis resulted in EHBP1 and EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization in endothelial cells. Overall, we demonstrate that EHBP1 and EHD2 regulate Dll4 endocytosis by anchoring caveolar endocytic pits to the actin cytoskeleton.

cell biology↗