ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells
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eLife assessment
This fundamental study demonstrates that the two isoforms of the ARPC5 subunit (ARPC5 and ARPC5L) of the Arp2/3 complex have specific functions in regulating cytoplasmic and nuclear actin filament assembly in response to DNA replication stress and T cell receptor signaling in T lymphocytes. The data presented in the manuscript are convincing and of good technical quality, and the study provides interesting new insights into specific cellular roles of different Arp2/3 isoforms in T lymphocytes.
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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.
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eLife assessment
This fundamental study demonstrates that the two isoforms of the ARPC5 subunit (ARPC5 and ARPC5L) of the Arp2/3 complex have specific functions in regulating cytoplasmic and nuclear actin filament assembly in response to DNA replication stress and T cell receptor signaling in T lymphocytes. The data presented in the manuscript are convincing and of good technical quality, and the study provides interesting new insights into specific cellular roles of different Arp2/3 isoforms in T lymphocytes.
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Reviewer #1 (Public Review):
Here the authors show using T cells that nuclear and cytoplasmic actin polymerization is differentially mediated by distinct Arp2/3 complexes containing ARPC5L or ARPC5. They further show that nuclear F-actin formation in T cells differs during calcium-mediated signaling versus replication stress response. They also provide information on molecular players mediating these 2 responses in T cells.
All in all this is an interesting study and provides valuable insights into the growing evidence of nuclear actin polymerisation for cell physiology and further highlights an intriguing importance of the composition of a given Arp2/3 complex via incorporation of different Arp subunits.
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Reviewer #2 (Public Review):
The authors use Jurkat CD4 T-cells stimulated with either antigen (via B-cells or immobilised) or using ionomycin and PMA to broadly stimulate as a model for T-cell activation. They have previously used this system to show that nuclear actin controls expression of some cytokines during T-cell activation. They describe a burst of actin assembly in the nucleus, followed by cytoplasmic actin assembly and organisation into an actin ring synapse in the case of the B-cell stimulation. The main novel observation is that knockdown of either ARPC5 or ARPC5L subunits of the Arp2/3 complex give different impairment of nuclear vs cytoplasmic actin assembly depending on the stimulus. The data are mostly clear and convincing and seem to be appropriately analysed. This study raises the interesting point that signal-induced …
Reviewer #2 (Public Review):
The authors use Jurkat CD4 T-cells stimulated with either antigen (via B-cells or immobilised) or using ionomycin and PMA to broadly stimulate as a model for T-cell activation. They have previously used this system to show that nuclear actin controls expression of some cytokines during T-cell activation. They describe a burst of actin assembly in the nucleus, followed by cytoplasmic actin assembly and organisation into an actin ring synapse in the case of the B-cell stimulation. The main novel observation is that knockdown of either ARPC5 or ARPC5L subunits of the Arp2/3 complex give different impairment of nuclear vs cytoplasmic actin assembly depending on the stimulus. The data are mostly clear and convincing and seem to be appropriately analysed. This study raises the interesting point that signal-induced actin assembly might use different isoforms of Arp2/3 complex depending on the context. These observations are of interest and reveal potential signal-dependent functions of the Arp2/3 subunits, but the study doesn't reveal a biological importance of these differences (e.g. consequences for gene expression or signaling) or explain how/why the different ARPC5 subunits can have different functions.
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Reviewer #3 (Public Review):
This study provides interesting new information on the cellular roles of different Arp2/3 complex isoforms. Previous studies have shown that T cell receptor signaling induces both cytoplasmic and nuclear actin filament assembly, which are dependent on the Arp2/3 complex. Moreover, nuclear actin filament assembly can be induced in response to DNA damage by the Arp2/3 complex. However, the possible roles of different Arp2/3 isoforms in these processes have not been reported. Here, Sadhu et al. demonstrate that the two isoforms of the ARPC5 subunit (ARPC5 and ARPC5L) have specific functions in these processes. By using knockdown and knockout cells, they provide evidence that cytoplasmic actin polymerization induced by T cell receptor activation is dependent on the ARPC5 isoform, whereas consequent nuclear actin …
Reviewer #3 (Public Review):
This study provides interesting new information on the cellular roles of different Arp2/3 complex isoforms. Previous studies have shown that T cell receptor signaling induces both cytoplasmic and nuclear actin filament assembly, which are dependent on the Arp2/3 complex. Moreover, nuclear actin filament assembly can be induced in response to DNA damage by the Arp2/3 complex. However, the possible roles of different Arp2/3 isoforms in these processes have not been reported. Here, Sadhu et al. demonstrate that the two isoforms of the ARPC5 subunit (ARPC5 and ARPC5L) have specific functions in these processes. By using knockdown and knockout cells, they provide evidence that cytoplasmic actin polymerization induced by T cell receptor activation is dependent on the ARPC5 isoform, whereas consequent nuclear actin filament assembly relies on the ARPC5L isoform. Interestingly, the nuclear actin polymerization induced by DNA replication stress is dependent on ARPC5. The authors also examined the upstream signaling pathways, and provide evidence that nuclear calcium-calmodulin signaling and N-WASP are specific activators of ARPC5L containing complexes.
Majority of the data presented in the manuscript appear of good technical quality, and the study provides interesting new insights into specific cellular roles of different Arp2/3 isoforms in T lymphocytes.
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