Differential N-terminal processing of beta and gamma actin in vivo

Actin is one of the most essential and abundant intracellular proteins, playing an essential physiological role as the major constituent of the actin cytoskeleton. Two cytoplasmic actins, beta- and gamma-actin, are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-terminus, making it very difficult to dissect their individual regulation in vivo. The majority of actins are N-terminally acetylated, following the removal of N-terminal Met. Here, we analyzed beta and gamma cytoplasmic actin N-termini in vivo and found that beta actin, unlike gamma actin, specifically undergoes sequential removal of N-terminal amino acid Asp residues. This processing affects ∼1-3% of beta actin in different cell types. We identified candidate enzymes capable of mediating this type of processing, and used CRISPR/Cas-9 to delete them, individually or together, in mammalian cell lines. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration, suggesting that the beta actin processing mediated by these enzymes is physiologically important to beta actin function. We propose that selective N-terminal processing of beta actin by sequential removal of Asp contributes to differentiating the functions of non-muscle actin isoforms in vivo.

Formation of Cytoplasmic Actin-Cofilin Rods is Triggered by Metabolic Stress and Changes in Cellular pH

Actin dynamics plays a crucial role in regulating essential cell functions and thereby is largely responsible to a considerable extent for cellular energy consumption. Certain pathological conditions in humans, like neurological disorders such as Alzheimer’s disease or amyotrophic lateral sclerosis (ALS) as well as variants of nemaline myopathy are associated with cytoskeletal abnormalities, so-called actin-cofilin rods. Actin-cofilin rods are aggregates consisting mainly of actin and cofilin, which are formed as a result of cellular stress and thereby help to ensure the survival of cells under unfavorable conditions. We have used Dictyostelium discoideum, an established model system for cytoskeletal research to study formation and principles of cytoplasmic actin rod assembly in response to energy depletion. Experimentally, depletion of ATP was provoked by addition of either sodium azide, dinitrophenol, or 2-deoxy-glucose, and the formation of rod assembly was recorded by live-cell imaging. Furthermore, we show that hyperosmotic shock induces actin-cofilin rods, and that a drop in the intracellular pH accompanies this condition. Our data reveal that acidification of the cytoplasm can induce the formation of actin-cofilin rods to varying degrees and suggest that a local reduction in cellular pH may be a cause for the formation of cytoplasmic rods. We hypothesize that local phase separation mechanistically triggers the assembly of actin-cofilin rods and thereby influences the material properties of actin structures.

Effects of Low and High Aneurysmal Wall Shear Stress on Endothelial Cell Behavior: Differences and Similarities

Background: Intracranial aneurysms (IAs) result from abnormal enlargement of the arterial lumen. IAs are mostly quiescent and asymptomatic, but their rupture leads to severe brain damage or death. As the evolution of IAs is hard to predict and intricates medical decision, it is essential to improve our understanding of their pathophysiology. Wall shear stress (WSS) is proposed to influence IA growth and rupture. In this study, we investigated the effects of low and supra-high aneurysmal WSS on endothelial cells (ECs).Methods: Porcine arterial ECs were exposed for 48 h to defined levels of shear stress (2, 30, or 80 dyne/cm2) using an Ibidi flow apparatus. Immunostaining for CD31 or γ-cytoplasmic actin was performed to outline cell borders or to determine cell architecture. Geometry measurements (cell orientation, area, circularity and aspect ratio) were performed on confocal microscopy images. mRNA was extracted for RNAseq analysis.Results: ECs exposed to low or supra-high aneurysmal WSS were more circular and had a lower aspect ratio than cells exposed to physiological flow. Furthermore, they lost the alignment in the direction of flow observed under physiological conditions. The effects of low WSS on differential gene expression were stronger than those of supra-high WSS. Gene set enrichment analysis highlighted that extracellular matrix proteins, cytoskeletal proteins and more particularly the actin protein family were among the protein classes the most affected by shear stress. Interestingly, most genes showed an opposite regulation under both types of aneurysmal WSS. Immunostainings for γ-cytoplasmic actin suggested a different organization of this cytoskeletal protein between ECs exposed to physiological and both types of aneurysmal WSS.Conclusion: Under both aneurysmal low and supra-high WSS the typical arterial EC morphology molds to a more spherical shape. Whereas low WSS down-regulates the expression of cytoskeletal-related proteins and up-regulates extracellular matrix proteins, supra-high WSS induces opposite changes in gene expression of these protein classes. The differential regulation in EC gene expression observed under various WSS translate into a different organization of the ECs’ architecture. This adaptation of ECs to different aneurysmal WSS conditions may affect vascular remodeling in IAs.

Abstract P485: Loss Of Acta2 In Cardiac Fibroblasts Does Not Prevent The Myofibroblast Differentiation Or Affect The Cardiac Repair After Myocardial Infarction

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair in the infarcted area. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2 / SMαA in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair remained largely unknown. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal survival rate after MI. Moreover, Acta2 deletion did not affect the function or overall histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT and Acta2 -null cardiac myofibroblasts. It was identified that Acta2 -null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a unique compensatory increase in the transcription level of Actg2 and an increase in the protein level of sarcomeric actin isoform(s). In addition, the specific muscle actin isoforms that were upregulated in Acta2 -null cardiac myofibroblasts varied between individual cells. Moreover, the formation of stress fibers by cytoplasmic actin isoforms, especially the cytoplasmic gamma-actin, was enhanced in Acta2 -null cardiac myofibroblasts despite their unchanged RNA and protein expression. In conclusion, the deletion of Acta2 does not prevent the myofibroblast differentiation of cardiac fibroblasts or affect the post-MI cardiac repair, and the increased expression and stress fiber formation of non-SMαA actin isoforms and the functional redundancy between actin isoforms are able to compensate for the loss of Acta2 in cardiac myofibroblasts.

Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage

Upon induction of DNA damage with 405 nm laser light, proteins involved in Base Excision Repair (BER) are recruited to DNA lesions. We find that the dynamics of factors typical of either short-patch (XRCC1) or long-patch (PCNA) BER are altered by chemicals that perturb actin or tubulin polymerization in human cells. Whereas the destabilization of actin filaments by Latrunculin B, Cytochalasin B or Jasplakinolide decreases BER factor accumulation at laser-induced damage, inhibition of tubulin polymerization by Nocodazole increases it. We detect no recruitment of actin to sites of laser-induced DNA damage, yet the depolymerization of cytoplasmic actin filaments elevates both actin and tubulin signals in the nucleus. While published evidence suggested a positive role for F-actin in double-strand break repair in mammals, the enrichment of actin in budding yeast nuclei interferes with BER, augmenting sensitivity to Zeocin. Our quantitative imaging results suggest that the depolymerization of cytoplasmic actin may compromise BER efficiency in mammals not only due to elevated levels of nuclear actin, but also of tubulin. Our study is one of few linking cytoskeletal integrity to BER. [Media: see text] [Media: see text]

Journey to the Center of the Cell: Cytoplasmic and Nuclear Actin in Immune Cell Functions

Actin cytoskeletal dynamics drive cellular shape changes, linking numerous cell functions to physiological and pathological cues. Mutations in actin regulators that are differentially expressed or enriched in immune cells cause severe human diseases known as primary immunodeficiencies underscoring the importance of efficienct actin remodeling in immune cell homeostasis. Here we discuss recent findings on how immune cells sense the mechanical properties of their environement. Moreover, while the organization and biochemical regulation of cytoplasmic actin have been extensively studied, nuclear actin reorganization is a rapidly emerging field that has only begun to be explored in immune cells. Based on the critical and multifaceted contributions of cytoplasmic actin in immune cell functionality, nuclear actin regulation is anticipated to have a large impact on our understanding of immune cell development and functionality.

Loss of Acta2 in cardiac fibroblasts does not affect myofibroblast differentiation or cardiac repair after myocardial infarction

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair in the infarcted area. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2 in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair were still not known. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal survival rate after MI. Moreover, Acta2 deletion did not affect the function or overall histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT cardiac fibroblasts and Acta2-null cardiac myofibroblasts. Additional analysis identified that Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a unique compensatory increase in the transcription level of Actg2 and a possible increase in the protein abundance of cytoplasmic actin isoforms. In conclusion, SMαA stress fibers are not required for myofibroblast differentiation of cardiac fibroblasts or the post-MI cardiac repair, and the increase in the expression of non-SMαA actin isoforms and the functional redundancy between actin isoforms are likely able to compensate for the loss of Acta2 in cardiac myofibroblasts.

Profilin2 regulates actin rod assembly in neuronal cells

Nuclear and cytoplasmic actin-cofilin rods are formed transiently under stress conditions to reduce actin filament turnover and ATP hydrolysis. The persistence of these structures has been implicated in disease pathology of several neurological disorders. Recently, the presence of actin rods has been discovered in Spinal Muscular Atrophy (SMA), a neurodegenerative disease affecting predominantly motoneurons leading to muscle weakness and atrophy. This finding underlined the importance of dysregulated actin dynamics in motoneuron loss in SMA. In this study, we characterized actin rods formed in a SMA cell culture model analyzing their composition by LC–MS-based proteomics. Besides actin and cofilin, we identified proteins involved in processes such as ubiquitination, translation or protein folding to be bound to actin rods. This suggests their sequestration to actin rods, thus impairing important cellular functions. Moreover, we showed the involvement of the cytoskeletal protein profilin2 and its upstream effectors RhoA/ROCK in actin rod assembly in SMA. These findings implicate that the formation of actin rods exerts detrimental effects on motoneuron homeostasis by affecting actin dynamics and disturbing essential cellular pathways.