Angiogenesis and vascular remodeling are driven by extensive endothelial cell movements. Here, we present in vivo evidence that endothelial cell movements are associated with oscillating lamellipodia-like structures, which emerge from cell junctions in the direction of cell movements. High-resolution time-lapse imaging of these junction-based lamellipodia (JBL) shows dynamic and distinct deployment of junctional proteins, such as F-actin, VE-cadherin and ZO1, during JBL oscillations. Upon initiation, F-actin and VE-cadherin are broadly distributed within JBL, whereas ZO1 remains at cell junctions. Subsequently, a new junction is formed at the front of the JBL, which then merges with the proximal junction. Rac1 inhibition interferes with JBL oscillations and disrupts cell elongation—similar to a truncation in ve-cadherin preventing VE-cad/F-actin interaction. Taken together, our observations suggest an oscillating ratchet-like mechanism, which is used by endothelial cells to move over each other and thus provides the physical means for cell rearrangements.
Asymmetric subcellular localization of mRNA is a common cellular phenomenon that is thought to contribute to spatial gene regulation. In highly polar neurons, subcellular transcript localization and translation are thought to enhance cellular efficiency and timely responses to external cues. Although mRNA localization has been observed in many tissues and numerous examples of the functional importance of this process exist, we still lack a systematic understanding of how the transcript sorting machinery works in a sequence-specific manner. Here, we addressed these gaps by combining subcellular transcriptomics and rationally designed sequence libraries. We developed a massively parallel reporter assay (MPRA) for mRNA localization and tested ~50,000 sequences for their ability to drive RNA localization to neurites of neuronal cell lines. By scanning the 3'UTR of >300 genes we identified many previously unknown localization regions and mapped the localization potential of endogenous sequences. Our data suggest two ways the localization potential can be encoded in the 3'UTR: focused localization motifs and broadly encoded localization potential based on small contributions. We identified sequence motifs enriched in dendritically localized transcripts and tested the potential of these motifs to affect the localization behavior of an mRNA. This assay revealed sequence elements with the ability to bias localization towards neurite as well as soma. Depletion of RNA binding proteins predicted or experimentally shown to bind these motifs abolished the effect on localization, suggesting that these motifs act by recruiting specific RNA-binding proteins. Based on our dataset we developed machine learning models that accurately predict the localization behavior of novel sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native transcripts. Applying similar systematic high-throughput approaches to other cell types will open the door for a comparative perspective on RNA localization across tissues and reveal the commonalities and differences of this crucial regulatory mechanism.
Molecular programs initiating cell fate divergence (CFD) are difficult to identify. Current approaches usually compare cells long after CFD initiation, therefore missing molecular changes at its start. Ideally, single cells which differ in their CFD molecular program but are otherwise identical are compared early in CFD. This is possible in diverging sister cells, which were identical until their mother's division and thus differ mainly in CFD properties. In asymmetrically dividing cells, divergent daughter fates are prospectively committed during division, and diverging sisters can thus be identified at the CFD start. Using asymmetrically dividing blood stem cells, we developed a pipeline (trackSeq) for imaging, tracking, isolating and transcriptome sequencing of single cells. Their identities, kinship and histories are maintained throughout, massively improving molecular noise filtering and candidate identification. In addition to many identified blood stem CFD regulators, we here provide this pipeline for use also in CFDs other than asymmetric division.
Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveal distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tri-cellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function during junction formation and stabilization during sprouting angiogenesis.
Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a 1VE-cadherin/F-actin based oscillatory ratchet mechanism 2 3 4 5 6 Abstract 21 22Angiogenesis and vascular remodeling are driven by a wide range of endothelial cell 23 behaviors, such as cell divisions, cell movements, cell shape and polarity changes. To 24 decipher the cellular and molecular mechanism of cell movements, we have analyzed 25 the dynamics of different junctional components during blood vessel anastomosis in 26 vivo. We show that endothelial cell movements are associated with oscillating 27 lamellipodia-like structures, which are orientated in the direction of these movements. 28These structures emerge from endothelial cell junctions and we thus call them junction-29 based lamellipodia (JBL). High-resolution time-lapse imaging shows that JBL are 30 formed by F-actin based protrusions at the front end of moving cells. These protrusions 31 also contain diffusely distributed VE-cadherin, whereas the junctional protein ZO-1 32 (Zona occludens 1) remains at the junction. Subsequently, a new junction is formed at 33 the front of the JBL and the proximal junction is pulled towards the newly established 34 distal junction. JBL function is highly dependent on F-actin dynamics. Inhibition of F-35 actin polymerization prevents JBL formation, whereas Rac-1 inhibition interferes with 36 JBL oscillations. Both interventions disrupt endothelial junction formation and cell 37 elongation. To examine the role of VE-cadherin (encoded by cdh5 gene) in this process, 38we generated a targeted mutation in VE-cadherin gene (cdh5 ubs25 ), which prevents VE-39 cad/F-actin interaction. Although homozygous ve-cadherin mutants form JBL, these 40 JBL are less dynamic and do not promote endothelial cell elongation. Taken together, 41 our observations suggest a novel oscillating ratchet-like mechanism, which is used by 42 endothelial cells to move along or over each other and thus provides the physical means 43 for cell rearrangements. 44 45 46 Introduction 47 Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, 48 which ensure proper organ assembly and function. The cardiovascular system is one of 49 the most ramified vertebrate organs and is characterized by an extraordinary plasticity. 50 It forms during early embryonic development, and it expands and remodels to adapt to 51 the needs of the growing embryo. In adult life, this plasticity allows flexible responses, 52 for example, during inflammation and wound healing 1,2 . 53 54 At the cellular level, blood vessel morphogenesis and remodeling are accomplished by 55 endothelial cell behaviors including cell migration, cell rearrangement and cell shape 56 changes 3-5 . This repertoire of dynamic behaviors allows endothelial cells to rapidly 57 respond to different contextual cues, for example during angiogenic sprouting, 58anastomosis, diapedesis or regeneration. In particular, it has been shown that 59 endothelial cells are very motile, not only during sprouting, but ...
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