Nuclear movement and positioning can be mediated by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which consist of Sad1/UNC-84 (SUN) proteins and Klarsicht/ANC-1/Syne Homology (KASH) proteins. KASH proteins bind SUN proteins via a short KASH domain. Unlike animal KASH domains, plant KASH domains are shorter, lack ability to form disulfide bonds and have a different, C-terminal sequence motif. We have examined the specificity of KASH domains using two Arabidopsis KASH proteins, WIP1 and SINE1. We show experimentally that the SINE1 KASH domain is required for SINE1 function in stomata and the WIP1 KASH domain for WIP1 function in root hairs, but that the SINE1 and WIP1 KASH domains are interchangeable for the WIP1 role in nuclear movement in pollen tubes. Through molecular modeling we found that SINE1 has two distinct binding modes depending on its interaction partner, SUN1 or SUN2, while WIP1 binds very similarly to both SUN1 and SUN2. We propose that this requirement for specific KASH domains reflects differences between the SUN1-SINE1 and SUN1-WIP1 interaction models that might indicate a different tolerance of the interactions to force.
Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.
Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.
Background Chimeric antigen receptor (CAR) T-cell therapy has demonstrated safety and modest efficacy against diffuse midline gliomas (DMGs), a highly aggressive pediatric brain tumor. However, mechanisms of CAR T-cell resistance in DMG settings remain unknown.Methods We compared the efficacy of B7-H3 CAR T-cells between SJ-DIPGX7c (DMG) and U87-MG (adult glioblastoma) patient-derived cell lines and showed impaired efficacy both in vitro and in vivo. We performed live-cell imaging and single-cell RNA sequencing to investigate deficiencies in immune synapse (IS) formation between CAR T-cells and DMGs. Lastly, we genetically deleted RASA2, a negative regulator of T cell activation, and evaluated the resulting impact on IS formation and quality, as well as in vitro and in vivo functionality.Results We show that limited efficacy of B7-H3 CAR T-cells is due to DMG-mediated inefficient interaction between CAR T-cells and DMG cells. Specifically, DMG cells impair the IS formation, resulting in poor CAR T-cell activation, cytokine secretion, and limited anti-tumor response in vivo. RASA2 deletion improved CAR T-cell activation through the formation of a more functional IS. RASA2-deleted CAR T-cells exhibited enhanced calcium flux, increased accumulation of activated signaling molecules and lytic granules at the synapse, and increased actin cytoskeleton dynamics, which produced larger synaptic areas and resulted in enhanced migration ex vivo. Further, RASA2-deleted CAR T-cells demonstrated improved in vitro functionality and superior early in vivo anti-tumor responses against DMGs compared with controls.Conclusions Our study highlights the importance of understanding tumor-specific factors that limit CAR T-cell response and using this information to design superior next-generation CAR T-cells. Specifically, we identify cytoskeleton remodeling and T cell motility as therapeutically actionable targets for future engineering approaches.
Rxiv cytoskeleton
Rxiv mechanobio