ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

Abstract

CD4 T cell activation induces nuclear and cytoplasmic actin polymerization via the Arp2/3 complex to activate cytokine expression and strengthen T cell receptor (TCR) signaling. Actin polymerization dynamics and filament morphology differ between nucleus and cytoplasm. However, it is unclear how the Arp2/3 complex mediates distinct nuclear and cytoplasmic actin polymerization in response to a common stimulus. In humans, the ARP3, ARPC1, and ARPC5 subunits of the Arp2/3 complex exist as two different isoforms, resulting in complexes with different properties. Here, we show that the Arp2/3 subunit isoforms ARPC5 and ARPC5L play a central role in coordinating distinct actin polymerization events in CD4 T cells. While ARPC5L is heterogeneously expressed in individual CD4 T cells, it specifically drives nuclear actin polymerization upon T cell activation. In contrast, ARPC5 is evenly expressed in CD4 T cell populations and is required for cytoplasmic actin dynamics. Interestingly, nuclear actin polymerization triggered by a different stimulus, DNA replication stress, specifically requires ARPC5 but not ARPC5L. TCR signaling but not DNA replication stress induces nuclear actin polymerization via nuclear calcium-calmodulin signaling and N-WASP. Diversity in the molecular properties and individual expression patterns of ARPC5 subunit isoforms thus tailors Arp2/3-mediated actin polymerization to different physiological stimuli.

Arp2/3 complex-dependent actin regulation protects the survival of tissue-resident mast cells

“…The reported defects, resulting in combined immunodeficiencies with severe inflammation and early-onset autoimmunity, inflammation, and mortality, highlight the clinical relevance of Arp2/3 complex dysfunction (64)(65)(66). Notably, isoform diversity within the Arp2/3 complex may determine cellspecific phenotypes (67,68), prompting further exploration of individual ARPC isoforms for MC biology.…”

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression

“…Actin remodeling in response to T cell receptor (TCR) engagement was monitored by forming circumferential F-actin rings as previously described [ 61 , 62 ]. In brief, stimulatory coverslips were prepared by coating with a 0.01% poly-L-lysine (PLL; Sigma) solution for 10 minutes at room temperature, followed by wet-chamber incubation for 3 hours at 37°C with 7 μg/ml anti-CD3 antibody (50 μl per coverslip, clone HIT3a against CD3E; BD Biosciences) in phosphate-buffered saline (PBS).…”

“…CYTOR RNA levels were normalized to the gapdh gene. Although we are aware that GAPDH expression is elevated following TCR stimulation, we did analyze several cellular genes in search of a better marker, but concluded that, e.g., genes for actin polymerization machinery are all affected more strongly by T cell activation than gapdh [ 62 ]. We therefore used gapdh for normalization.…”

A General Method to Accurately Count Molecular Complexes and Determine the Degree of Labelling in Cells Using Protein Tags

“…To address this, we first used ProDOL in combination with CoPS to determine the absolute SLP-76 copy number in response to TCR engagement in the absence of presence of Nef. To this end, Jurkat CD4 T cells stably expressing a SLP-76-HaloTag fusion protein were labelled in suspension with Halo-SiR and stimulated on glass cover slides functionalised with -CD3 antibodies for activation [28][29][30] . To synchronise the activation reaction of individual cells, only cells that adhered to the cover slide within the first 3 seconds were observed and all other cells washed away.…”