Fig 2: Differential involvement of class I nucleation-promoting factors (NPFs) in T cell activation and replication stress-mediated nuclear F-actin (NFA) formation.(A) Representative immunoblots show knockout (KO) of NWASP, WASHC5, and WAVE2 class I NPFs, respectively, in JNLA cells. Identical amounts of cell lysates were loaded per lane and signal intensity judged based on the loading control (GAPDH or Tubulin). Black arrowheads or asterisks mark specific and unspecific bands, respectively, as identified based on signal reduction in the KO cell line. The numbers indicated below the respective blots represent the mean ± SD values from three independent experiments, based on the densitometric quantification of the bands, normalized to the loading control and compared to the non-targeting control (NTC) protein levels (which is set to 1). (B) Representative spinning-disk confocal still images (maximum projection) of JNLA cells with indicated NPF KO showing NFA formation post activation with PMA/ionomycin (P/I). Movies were acquired for 5 min post PI addition with acquisition every 30 s. Arrows point to the NFA. Scale bar, 3 µm. (C) Quantification of NFA formation in respective NPF KO cells relative to the NTC-treated cells. Each data point indicates the mean value of an independent experiments with 30 analyzed per condition with the indicated mean ± SD from three independent experiments. (D) Representative confocal images indicate averaged intensity projections of Phalloidin-647-stained F-actin ring (AR) formation in fixed/permeabilized JNLA cells with respective NPF KO upon activation on coverslips coated with anti-CD3+CD28 antibodies. Arrowheads point to the AR at the PM upon TCR activation, and the dotted box in white shows the cell in zoomed view in the inset (top right). Scale bar, 5 µm. (E) Quantification of Phalloidin-stained AR formation in KO cells relative to the NTC-treated cells. Each data point indicates the mean value of an independent experiment with at least 100 cells analyzed per condition with indicated mean ± SD from three independent experiments. (F) Shown are representative spinning-disk confocal still images (maximum projection) of the aphidicolin (APH)-treated NPF KO or control cells, respectively. The movies were acquired for 5 hr with acquisition every 15 min post pretreatment of cells with APH. The stills at the indicated time points are representative of the time point where the maximal NFA burst has been observed in each condition. (G) Quantification of the % of cells with NFA bursts (within first 2 hr of imaging) post replication stress induction in control and KO cells. Each data point indicates the mean value of an independent experiment with 40–60 cells analyzed per condition with indicated mean ± SD from three independent experiments. One-way ANOVA with Kruskal–Wallis test was used to determine statistical significances, where *p=0.0332 and ns: not significant. Also see related Figure 7—figure supplement 1A-F. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8.

Fig 3: Cellular distribution of ARPC5 isoforms.(A) Shown are representative spinning-disk confocal images (maximum projection) of ARPC5.mCherry and ARPC5L.mCherry distribution in unstimulated JNLA cells. White arrows point to the respective C5 or C5L punctae seen in the nucleus. Also see related Figure 5—figure supplement 1A and B. (B) Subcellular distribution of the ARPC5 subunit and its isoform ARPC5L was determined by biochemical fractionation of JNLA cells with respective knockout of either C5 or C5L in the bulk culture. Representative immunoblots reveal levels of ARPC5 isoforms in the whole cell extract (WCE), cytoplasmic (C), and nuclear (N) fractions in the indicated JNLA knockout cells post fractionation. GAPDH and hnRNPL were used as markers for cytoplasmic and nuclear compartments, respectively. (C) Representative, deconvoluted, and segmented stimulated emission depletion (STED) single-plane images show endogenous nuclear actin filaments (stained with Phalloidin-647N) and ARPC5.mCherry/ARPC5L.mCherry (signal enhanced with anti-mCherry with secondary antibody in atto-594 channel) in A3.01 T cells, stimulated with PMA/ionomycin (P/I) for 30 s. Arrows (in white) point to the colocalization events. Percent colocalization is mentioned as mean ± SD (in white bar, top right) for each of the isoforms from three independent experiments. Scale bar, 500 nm. (D) The dot plot shows the frequency of colocalization of ARPC5 and ARPC5L with nuclear F-actin (from representative STED-deconvolved and segmented super-resolved images shown in Figure 5C) in A3.01 cells post 30 s of stimulation with PMA + ionomycin. Each dot represents colocalization events per cell that was analyzed. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8.

Fig 4: ARPC5 isoforms differentially regulate nuclear and plasma membrane actin polymerization.(A) Representative immunoblots show knockdown of ARPC1 and ARPC5 isoforms in bulk JNLA cells treated with indicated shRNAs. Black arrowheads indicate the specific bands, and black asterisks mark unspecific bands. Note that the ARPC5L antibody also detects ARPC5 (marked by red asterisk). The numbers indicated below the respective blots represent the mean ± SD values from four independent experiments, based on the densitometric quantification of the bands, normalized to GAPDH and compared to the non-targeting control (NTC) protein levels (which is set to 1). (B) Representative spinning-disk confocal still images of JNLA cells treated with indicated shRNA show stills post activation with PMA/ionomycin (P/I). Arrows point to the nuclear F-actin (NFA). Relates to Figure 3—figure supplement 1A,B. Scale bar, 3 µm. (C) Quantification of NFA formation in shRNA-treated cells relative to the scrambled control-treated cells. Mean ± SD of four independent experiments where 30 cells were analyzed per condition per experiment. Each dot represents the mean of each independent experiment. (D) Representative immunofluorescence images indicate averaged intensity projections of Phalloidin-647-stained F-actin ring (AR) formation in JNLA cells treated with indicated shRNA upon activation on coverslips coated with anti-CD3+CD28 antibodies. Arrowheads point to the F-actin ring at the PM. (E) Quantification of Phalloidin-stained AR formation in shRNA-treated cells relative to the control-treated cells. In (C, E), each data point indicates the mean value of an independent experiment consisting of two technical replicates with at least 100 cells analyzed per condition with indicated mean ± SD from four independent experiments. One-way ANOVA with Kruskal–Wallis test was used to determine statistical significances, where *p=0.0332, **p=0.0021, and ns: not significant. Scale bar, 5 µm. See also related Figure 3—figure supplement 1C-F. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8.