Using with the proteomic and microscopy approach, we find that the combination of cyclic hypertensive pressure stimulation and matrix compliance in extracellular microenvironment is necessary to give rise to a full phenotypic switch of vascular smooth muscle cell, including significant changes in atherosclerosis-associated protein expression, cytoskeletal organization, and the formation of podosome. The underlying principle of mechanosensitive response is orchestrated by cofilin. Specifically, unphosphorylated cofilin plays the critical role in triggering actin nucleation. Pressure stimuli initiates the calcium-dependent slingshot activation that dephosphorylates cofilin, while the reduction of substratum stiffness suppresses the signal cascade of RhoA-ROCK2-LIMK2 and restrains cofilin phosphorylation. Collectively, the decrease of cofilin phosphorylation promote actin nucleation, and the fast oscillatory nature of podosome assembly act as the feedback loop to spatiotemporally regulate RhoA activities needed for cell migration. For more details, see Swiatlowska et al., Science Advances 2022.

The dynamic distribution of phosphoinositide lipids orches- trates intracellular compartmentalization and protein sorting. Here, we report the PI(3,4)P2-dependent endocytosis of adhe- sion receptor integrin-beta3 at the invadopodium and its im- plication in promoting invasive cell migration. PI(3,4)P2-rich compartment in the plasma membrane contains integrin-beta3 and is actively internalized through SNX9-mediated membrane invagination as well as dynein-mediated membrane tubulation along cortical microtubule tracks. Accelerated integrin endo- cytosis promotes adhesion turnover, and subsequent integrin exocytosis further supports cell migration. In general, the functional link between PI(3,4)P2 biogenesis and membrane internalization can be applicable to other membrane molecules at the invadopodium and provide insights into the regulation of cell migration. For more details, see Zhen and Yu, PNAS 2021.

DNA double-strand breaks (DSBs) are arguably the most toxic form of DNA damage. In this study, we report a role of the DYRK1B kinase as a suppressor of ribosomal DNA (rDNA) transcription during cellular responses to rDNA DSBs. We found that DYRK1B is targeted to laser-induced DNA damage sites within the nucleolar compartment, and is required of pausing of local transcriptional activities. Failing to suppress rDNA transcription resulted in sustained DNA damage and elevated frequencies of chromosomal aberrations, which in turn led to loss of rDNA copy number and cell hypersensitivity to rDNA DSBs. Together, our findings establish DYRK1B as a component of the mammalian nucleolar DSB response network. For more details, see Dong et al., NAR 2021.

Membrane signals to trigger actin polymerization at the macrophage podosome are poorly understood. Here, we report that PI(3,4,5)P3 lipids serve as the key membrane messenger. Local enrichment of PI(3,4,5)P3 lipids by PIK3CB activates WASP-mediated F-actin polymerization and initiate macrophage podosome formation. WASP-5KE mutant lacks the membrane association, and blockage of PIK3CB suppresses the podosome assembly. We further investigate the upstream signaling pathway and reveal that Abl1 and Src family kinase (Src/Hck) are essential for PIK3CB activation. In particular, Y488 phosphorylated Abl1 by Src and Hck acts as the key element to recruitment of PIK3R1 and promote podosome formation. Knockdown or chemical inhibition of Src/Abl1/PIK3CB signaling axis strongly blocks the podosome formation, suppress matrix degradation, and impede chemotactic migration of macrophages. For more details, see Zhou et al., JCS 2020.

The mechanism of integrin endocytosis and adhesion turnover at the podosome is largely unknown. Different from contractile forces at focal adhesion, protrusive F-actin polymerization at the podosome can maneuver membrane curvature signals to promote the endocytosis of membrane receptors. Here, we find that integrin-β3 and RGD ligands are locally internalized at the podosome adhesion and then sorted into the endosome. We carefully follow the process of podosome formation and find that protrusive F-actin polymerization causes membrane deformation and N-BAR domain protein BIN1 recruitment at the podosome ring. BIN1 subsequently recruits dynamin2 to promote the endocytosis. Inhibitions of BIN1-dynamin2 association effectively block the endocytosis of RGD-integrin-β3. Thus, protrusive F-actin polymerization results in the membrane invagination and BIN1-dynamin2 dependent endocytosis of integrin-beta3 at the podosome ring. For more details, see Cao et al., Communications Biology 2020.

Membrane-bound myosin-1 is one of the important elements to convert lipid signaling and modulate F-actin cytoskeleton at the cell-matrix adhesions. Misregulated myosin-1s often cause malfunctions in F-actin assembly and phosphatidylinositol lipid signaling. Tail-less mutant MYO1E-Y695X becomes cytosolic and absent from the plasma membrane and causes structural defects in F-actin assembly. Here, we compare the axial distribution of 26 podosome core components and report that long-tailed myosin-1e (MYO1E) is enriched at the ventral layer of the podosome core in a PI(3,4,5)P3-dependent manner. The combination of TH1 and TH2 (TH12) of MYO1E tail domains contains the essential motif for PI(3,4,5)P3-dependent membrane association and ventral localization at the podosome. TH12 domain of MYO1E serves as a regulatory component to connect phosphatidylinositol signaling to F-actin polymerization at the podosome. For more details, see Zhang et al., MBoC 2019.

TRAIP mutations are associated with microcephalic primordial dwarfism and Seckel syndrome. TRAIP encodes a nucleolar protein that is a pivotal component of the mammalian replicative stress response. TRAIP migrates to UV-induced DNA lesions via a direct interaction with the DNA replication clamp PCNA. Because TRAIP concentrates in the nucleoli, delineating how UV triggers its release from the nucleoli is key to understanding its regulation in the protection of genome integrity, especially as this may represent a rate-limiting step that underlies its responsiveness to replicative stress. In this study, we utilize a combinational approach of photo-switching and photo-bleaching and provide several lines of evidence to support a two-step mechanism that orchestrates TRAIP trafficking. Our data suggest that TRAIP associates with R-loops in the nucleoli, and that its release is coupled to dampened rDNA synthesis and R-loop dissolution. For more details, see Chen et al., NAR 2018.

The turnover of integrin receptors is critical for cell migration and adhesion dynamics. We find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD and integrin-beta3. For more details, see Yu et al., Nature Communications 2015.

I developed nano-patterned supported membranes as a novel platform to decipher how mechanical cues in matrices regulate cell adhesion transformation. My recent report indicated that the formation of different adhesion structures, such as focal adhesion and podosome are modulated by mechanical characteristics of matrices. Lack of traction force at activated integrin clusters results in podosome formation in PI3K and FAK/PYK2 dependent manner. The switching between classic focal adhesions to macrophage-like podosomes is remarkable. This groundbreaking finding directly suggests programmable adhesion transformation by matrix-mediated mechano-transduction. For more details, see Yu et al., Cell reports, 2013.

To address the spatial-temporal regulation of integrin activation, I utilized self-assembling proteolipid bilayer membrane with fluorescent cyclic RGD peptides as adhesion substrates. This configuration, known as a supported membrane, preserves the important characteristics of cell membrane, i.e. two-dimensional mobility. Fluid supported membrane can also be physically partitioned by pre-fabricated nano-barriers and can passively modulate the spatial organization patterns of ligand-receptor complexes. Combined with state-of-art fluorescence microscopy, it enables me to study the activation of integrin receptors and dynamical assembly of functional complexes during early adhesion formation. For more details, see Yu et al., PNAS 2011.