A mechanism with severing near barbed ends andannealing explains structure and dynamics of dendriticactin networks
- Lehigh University, United States
- Kyoto University, Japan
Abstract
Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/- 35𝑜 orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology.
Data availability
All data reported in this project are present within the published figures and Supplemental Information. The code for simulations is available at https://github.com/vavylonis/LamellipodiumSeverAnnealand will allow for all simulation plots to be reproduced. The experimental SiMS data of Figure 6 and Figure 6-supplement 1 have been provided as excel files containing the speckle tracks using the SpeckleTrackerJ ImageJ plugin
Article and author information
Author details
Dimitrios Vavylonis
Funding
National Institutes of Health (R35GM136372)
- Danielle Holz
- Aaron R Hall
- Eiji Usukura
- Sawako Yamashiro
- Naoki Watanabe
- Dimitrios Vavylonis
National Institutes of Health (R01GM114201)
- Danielle Holz
- Aaron R Hall
- Dimitrios Vavylonis
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Holz et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A mechanism with severing near barbed ends andannealing explains structure and dynamics of dendriticactin networks
eLife 11:e69031.
https://doi.org/10.7554/eLife.69031
Further reading
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- Cell Biology
Distal appendages are ninefold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for the formation of the primary cilium, by regulating at least four critical steps: preciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here, we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in the RAB34+ vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
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- Cell Biology
- Medicine
Background:
Pulmonary vascular remodeling is a progressive pathological process characterized by functional alterations within pulmonary artery smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs). Mechanisms driving the transition to a diseased phenotype remain elusive.
Methods:
We combined transcriptomic and proteomic profiling with phenotypic characterization of source-matched cells from healthy controls and individuals with idiopathic pulmonary arterial hypertension (IPAH). Bidirectional cellular crosstalk was examined using direct and indirect co-culture models, and phenotypic responses were assessed via transcriptome analysis.
Results:
PASMC and PAAF undergo distinct phenotypic shifts during pulmonary vascular remodeling, with limited shared features, such as reduced mitochondrial content and hyperpolarization. IPAH-PASMC exhibit increased glycosaminoglycan production and downregulation of contractile machinery, while IPAH-PAAF display a hyperproliferative phenotype. We identified alterations in extracellular matrix components, including laminin and collagen, alongside pentraxin-3 and hepatocyte growth factor, as potential regulators of PASMC phenotypic transitions mediated by PAAF.
Conclusions:
While PASMCs and PAAFs retain their core cellular identities, they acquire distinct disease-associated states. These findings provide new insights into the dynamic interplay of pulmonary vascular mesenchymal cells in disease pathogenesis.
Funding:
This work was supported by Cardio-Pulmonary Institute EXC 2026 390649896 (GK) and Austrian Science Fund (FWF) grant I 4651-B (SC).
