DataSheet8_Interaction of LARP4 to filamin A mechanosensing domain regulates cell migrations.ZIP

Filamin A (FLNA) is an actin cross-linking protein that mediates mechanotransduction. Force-dependent conformational changes of FLNA molecule expose cryptic binding site of FLNA, allowing interaction with partners such as integrin, smoothelin, and fimbacin. Here, we identified La-related protein 4 (LARP4) as a new FLNA mechanobinding partner. LARP4 specifically interacts with the cleft formed by C and D strands of immunoglobulin-like repeat 21 (R21) which is blocked by A strand of R20 without force. We validated the interaction between LARP4 and FLNA R21 both in vivo and in vitro. We also determined the critical amino acid that is responsible for the interaction and generated the non-FLNA-binding mutant LARP4 (F277A in human: F273A in mouse Larp4) that disrupts the interaction. Fluorescence recovery after photobleaching (FRAP) of GFP-labeled LARP4 in living cells demonstrated that mutant LARP4 diffuses faster than WT LARP4. Proximity ligation assay (PLA) also confirmed their interaction and disruption of actin polymerization diminishes the interaction. Data mining of RNAseq analysis of LARP4 knockdown (KD) HEK293T cells suggested that LARP4 is involved in morphogenesis and cell motility. Consistent with this prediction, we found that KD of LARP4 increases cell migration speed and expression of the F277A mutant LARP4 in LARP4-KD cells also leads to a higher cell migration speed compared to WT LARP4. These results demonstrated that the LARP4 interaction with FLNA regulates cell migration.

细丝蛋白A（Filamin A, FLNA）是一种介导机械转导的肌动蛋白交联蛋白。FLNA分子的力依赖性构象变化会暴露其隐蔽结合位点，使其能够与整合素（integrin）、smoothelin及fimbacin等结合伴侣相互作用。本研究中，我们鉴定出La相关蛋白4（La-related protein 4, LARP4）为新型FLNA机械结合伴侣。LARP4可特异性结合免疫球蛋白样重复序列21（immunoglobulin-like repeat 21, R21）的C、D链形成的裂隙；在无机械力的情况下，该裂隙会被R20的A链所遮蔽。我们分别在体内（in vivo）与体外（in vitro）验证了LARP4与FLNA R21的相互作用。此外，我们确定了介导该相互作用的关键氨基酸位点，并构建了无法结合FLNA的LARP4突变体（人类中为F277A，小鼠Larp4中为F273A），该突变体可阻断二者的相互作用。对活细胞中绿色荧光蛋白（green fluorescent protein, GFP）标记的LARP4开展荧光漂白后恢复实验（fluorescence recovery after photobleaching, FRAP），结果显示突变型LARP4的扩散速率快于野生型（wild type, WT）LARP4。邻近连接实验（proximity ligation assay, PLA）进一步证实了二者的相互作用，而肌动蛋白聚合受阻则会减弱该相互作用。对LARP4敲低（knockdown, KD）的HEK293T细胞进行RNA测序（RNA sequencing, RNAseq）数据分析后发现，LARP4参与细胞形态发生与细胞运动调控。与该预测一致，我们发现LARP4敲低可提升细胞迁移速率；在LARP4敲低细胞中表达F277A突变型LARP4，其细胞迁移速率同样高于表达野生型LARP4的细胞。以上结果表明，LARP4与FLNA的相互作用可调控细胞迁移。