Biology subjects

Hirosawa, K. M.

Publications and source records attributed to Hirosawa, K. M..

4 recordsLinked to original sources

bioRxiv2024

Single-molecule detection of transient dimerization of opioid receptors 1: Homodimers' effect on signaling and internalization

Opioid receptors (ORs) are critical for endogenous and synthetic analgesics. Their homodimerization is considered important for their pharmacological diversities, but whether they form homodimers remains controversial. Here, we established that the three classical ORs, -, {kappa}-, and {delta}-ORs (MOR, KOR, and DOR, respectively) undergo repeated transient (120-180 ms) homodimerizations every few seconds. This was done by using single-molecule imaging and developing theories for analyzing single-molecule colocalization data, which provide the key parameters, homodimer-monomer dissociation equilibrium constants and rate constants. Their 9-26 amino-acid C-terminal cytoplasmic domains, without sequence similarities, are involved in specific homodimerization, whereas the transmembrane domains provide less specific affinities. Using the membrane-permeable peptides mimicking the C-terminal homodimerization sequences which block homodimerizations, functions of monomers and homodimers were dissected. KOR and DOR homodimers, but not MOR homodimers, activate downstream G-proteins differently from monomers upon agonist addition, without influencing OR internalization. These findings could guide strategies to enhance OR-based analgesia.

biophysics↗

bioRxiv2024

Extracellular vesicles adhere to cells predominantly through the interaction of CD151-associated integrin heterodimers and GM1 with laminin

Tumor-derived extracellular vesicles (EVs) have attracted significant attention, yet the molecular mechanisms that govern their specific binding to recipient cells remain elusive. Our in vitro study utilizing single-particle tracking demonstrated that integrin heterodimers comprising 6{beta}4 and 6{beta}1 and ganglioside, GM1 are responsible for the binding of small-EV (sEV) subtypes to laminin. EVs derived from four distinct tumor cell lines, regardless of size, exhibited high binding affinities for laminin but not for fibronectin, although fibronectin receptors are abundant in EVs and have functional roles in EV-secreting cells. Our findings revealed that integrins in EVs bind to laminin via the conventional molecular interface, facilitated by CD151 rather than by inside-out signaling of talin-1 and kindlin-2. Super-resolution movie observation revealed that sEV integrins bind only to laminin on living recipient cells. Furthermore, sEVs bound to HUVEC and induced cell branching morphogenesis in a laminin-dependent manner. Thus, we demonstrated that EVs predominantly bind to laminin on recipient cells, which is indispensable for cell responses. SummaryQuantitative assessments using single-molecule imaging and super-resolution microscopy revealed that all extracellular vesicle subtypes derived from four distinct tumor cell lines, regardless of size, bind to laminin predominantly via CD151-facilitated integrin heterodimers and GM1, leading to response of recipient cells.

cell biology↗

bioRxiv2024

The uptake of small extracellular vesicles by recipient cells is facilitated by paracrine adhesion signaling

Small extracellular vesicles (sEVs) play crucial roles in intercellular communication. However, the internalization of individual sEVs by recipient cells has not yet been directly observed. Here, we examined these mechanisms using state-of-the-art imaging techniques. Single-molecule imaging showed that tumor-derived sEVs can be classified into several subtypes. Simultaneous single-sEV particle tracking and observation of super-resolution movies of membrane invaginations in living cells revealed that all sEV subtypes were internalized via clathrin-independent endocytosis mediated by galectin-3 and LAMP-2C, while some subtypes that recruited raft markers were internalized through caveolae. Integrin {beta}1 and talin-1 accumulated in recipient cell plasma membranes beneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggered Ca2+ mobilization induced by the activation of Src family kinases and PLC{gamma}. Subsequent Ca2+-induced activation of calcineurin-dynamin promoted sEV internalization, leading to the recycling pathway. Thus, we have clarified the detailed mechanisms of sEV internalization driven by paracrine adhesion signaling.

cell biology↗

bioRxiv2021

iTRVZ: liquid nano-platform for signal integration on the plasma membrane

Cell survival in immune-competent organisms relies on both growth and immune evasion, yet these processes have been studied as independent cellular programs. Here, using super-resolution single-molecule imaging, we identify GEM (Growth and Evasion Metastable hub), a {approx}34-nm, metastable, nano-liquid, plasma membrane hub that physically integrates receptor-type tyrosine kinase (RTK) inputs with CD59-mediated immune-evasion signaling within the same assembly. GEM is formed by LLPS-like protein clustering on PI(4,5)P2-containing raft-nanodomains. Upon concurrent RTK and CD59 stimulations, GEM recruits both receptors and downstream kinases, enabling reciprocal activation via nano-liquid confinement that supralinearly amplifies PLC{gamma}-IP3-Ca2+ and PI3K-Akt survival signaling outputs. In mice, disrupting GEM suppresses tumor growth in vivo, establishing nano-liquid GEM as an integrator that physically couples growth and CD59-mediated immune-evasion signaling for cell survival.

cell biology↗