Marcksl1 modulates endothelial cell mechanoresponse to haemodynamic forces to control blood vessel shape and size

Kondrychyn, Igor; Kelly, Douglas J.; Taberner, Núria; Nomori, Akane; Kato, Kagayaki; Chong, Jeronica; Nakajima, Hiroyuki; Okuda, Satoru; Mochizuki, Naoki; Phng, Li-Kun

doi:10.1038/s41467-020-19308-5

Cited by 31 publications

(35 citation statements)

References 53 publications

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“…Actin plays a pivotal role in angiogenesis both from a cell migration and vessel stabilization aspect [57][58][59][60][61][62]. Loss of normal actin architecture has been shown to drastically affect virtually all facets of blood vessel formation [63][64][65][66][67]. In this sense, our results are not surprising in that actin misregulation promoted such a profound effect on sprouting parameters.…”

Section: Discussionmentioning

confidence: 59%

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

Francis

Kincross

Kushner

2022

Preprint

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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.

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Section: Discussionmentioning

confidence: 59%

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

Francis

Kincross

Kushner

2022

Preprint

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“…Macrophage migration inhibitory factor (Mif), an inflammatory cytokine with chemokine functions that has been implicated in angiogenesis (Amin et al, 2003), was robustly expressed in E9.5 brain endothelial cells, but markedly downregulated in later stages. Marcksl1, a gene involved in blood vessel shape and size (Kondrychyn et al, 2020), was the most differentially upregulated gene in the E12.5 brain endothelium (Mif and Marcksl1 are labeled in purple, Figure 5E), while the amino acid transporter solute carrier transporter 7a5 (Slc7a5) (Tarlungeanu et al, 2016), as well as other blood brain barrier markers (denoted in red), initiated expression at E16.5 when blood brain barrier formation begins (Ben-Zvi et al, 2014;Hupe et al, 2017). Conversely, expression of plasmalemma vesicle-associated protein (Pvlap/ Mecca 32), a pan-endothelial marker that is lost in the mature BBB endothelium (Benz et al, 2019;Guo et al, 2016), was dramatically decreased after E12.5.…”

Section: Maturation Specific Regions Of Accessible Chromatin and Unique Transcription Factormentioning

confidence: 99%

Defining the Transcriptional and Epigenetic Basis of Organotypic Endothelial Diversity in the Developing and Adult Mouse

Gutierrez

Hill

Largoza

et al. 2021

Preprint

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Significant phenotypic differences exist between the vascular endothelium of different organs, including cell-cell junctions, paracellular fluid transport, shape, and mural cell coverage. These organ-specific morphological features ultimately manifest as different functional capacities, as demonstrated by the dramatic differences in capillary permeability between the leaky vessels of the liver compared to the almost impermeable vasculature found in the brain. While these morphological and functional differences have been long appreciated, the molecular basis of endothelial organ specialization remains unclear. To determine the epigenetic and transcriptional mechanisms driving this functional heterogeneity, we profiled accessible chromatin, as well as gene expression, in six different organs, across three distinct time points, during murine development and in adulthood. After identifying both common, and organ-specific DNA motif usage and transcriptional signatures, we then focused our studies on the endothelium of the central nervous system. Using single cell RNA-seq, we identified key gene regulatory networks governing brain blood vessel maturation, including TCF/LEF and FOX transcription factors. Critically, these unique regulatory regions and gene expression signatures are evolutionarily conserved in humans. Collectively, this work provides a valuable resource for identifying the transcriptional regulators controlling organ-specific endothelial specialization and provides novel insight into the gene regulatory networks governing the maturation and maintenance of the cerebrovasculature.

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“…This indicates that the endothelial lining of the cardioids is tightly controlled and still actively undergoes rearrangement at day 7 post-induction. 23 Contrarily, cardiomyocytes showed elevated levels of sarcomeric structural proteins MYL3, MYH6, TNNT2, TNNC1 and TNNI1 involved in muscle contraction. 20,21 Apart from well-known marker we also identified WDR1, an actin interacting protein, to be highly abundant in cardiomyocytes.…”

Section: Mainmentioning

confidence: 99%

An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity

Hartlmayr

Ctortecka

Seth³

et al. 2021

Preprint

105

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The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.

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Marcksl1 modulates endothelial cell mechanoresponse to haemodynamic forces to control blood vessel shape and size

Cited by 31 publications

References 53 publications

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

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

Defining the Transcriptional and Epigenetic Basis of Organotypic Endothelial Diversity in the Developing and Adult Mouse

An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity

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