Takanari Inoue

E-cadherin suppresses invasion and promotes metastasis in invasive ductal breast cancer, through suppression of reactive oxygen species (ROS). We sought to determine the cellular source of apoptosis-inducing ROS and to identify the domains of E-cadherin that are essential for cancer cell survival. Inhibition of mitochondria-derived ROS was sufficient to rescue cell death following E-cadherin deletion, in both 3D colony formation and in vivo metastasis assays. We next generated Cdh1 constructs by mutating amino acids or deleting domains, then tested their ability to rescue colony formation in E-cadherin–deleted cells. Surprisingly, E-cadherin constructs in which the extracellular domains were deleted, adhesion was compromised, or catenin interactions were blocked could rescue elevated mitochondrial ROS and 3D colony formation. Conversely, a construct lacking the cytoplasmic domain was unable to rescue ROS or cell death. Our findings reveal that E-cadherin’s ability to regulate mitochondrial ROS, cancer cell survival, and metastatic outgrowth is separable from its role in intercellular adhesion.

Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise induction of microtubule acetylation within minutes in live cells. Implementing optoTAT in migration assays, we observed striking and rapid responses at both molecular and cellular levels. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation, enabling a dynamic crosstalk between the actin and microtubule cytoskeletal networks in the coordination of cellular motility.

Microtubule (MT) bundling is a conserved organizational feature of the cytoskeleton that accompanies MT stabilization, suggesting that it is involved in diverse cellular processes, including mitosis, migration, and axon morphogenesis. Although microtubule-associated proteins are known to induce MT bundling, whether bundling itself is sufficient to alter MT properties and cellular behavior has remained difficult to address due to the lack of tools that selectively manipulate MT bundling in living cells. Here, we describe the development of a genetically encoded, protein-based “MT-Bundler” by coupling an MT-binding motif to a biologically inert oligomerization scaffold, enabling direct and tunable crosslinking of intracellular MTs. The expression of MT-Bundler consisting of MAP4 and Azami-Green, drove robust MT bundling, but also conferred marked resistance to depolymerization, and elevated MT acetylation. Functionally, enforced MT bundling disrupts cell division and migration and suppresses neurite and axon outgrowth. To confirm the causal relationship behind these findings, we further engineered chemically and optically inducible MT-Bundlers that permit rapid, reversible, and spatiotemporally precise control of MT bundling. Acute induction of MT bundling triggers a rapid increase in MT acetylation, revealing bundling as an upstream organizational cue that promotes luminal access of the acetyltransferase ATAT1. Notably, MT stabilization persists even in the absence of acetylation, demonstrating that bundling itself is sufficient to mechanically stabilize MTs. Together, these results identify MT bundling as a primary determinant of MT stability and modification, establishing MT-Bundlers as a versatile tool to dissect the mechanistic basis of MT bundling in living cells.

Cell migration is a fundamental biological process central to a number of physiological and pathophysiological events. Traditional genetic and pharmacological approaches have identified crucial molecular regulators of migration, yet they often lack the spatial and temporal resolution required to interrogate the highly dynamic signaling events that govern cell locomotion. Chemogenetic and optogenetic platforms—genetically encoded inducible systems activated by chemical or light stimuli, respectively—have emerged as powerful tools for achieving precise, on-demand control over protein function in living cells. These systems enable researchers to dissect molecular signaling pathways in real time and with subcellular precision, even as cells are actively migrating. Together with advances in de novo protein design, biosensors, and live-cell imaging, inducible molecular tools are transforming our ability to manipulate and elucidate the intricate mechanisms underlying cell motility. Looking forward, the application of these technologies in animal models will be crucial for gaining deeper physiological and pathophysiological insights.

Cilia are hair-like organelles that protrude from the cell surface. In mammals, tracheal multiciliated cells (MCCs) play an important role in elimination of hazardous microorganisms by driving a unidirectional mucus flow. Although uniform orientation of ciliary beating is critical for the unidirectional flow, it remains unknown how MCCs establish uniform orientations and maintain identities of hundreds of ciliary membranes. This study focuses on investigating the roles of Tubulin Polymerization Promoting Family Member 3 (Tppp3) in MCC function. We generated a Tppp3-deficient mouse ( Tppp3 ^{∆ex2-4/∆ex2-4} ; Tppp3 knockout (KO)) and found that the Tppp3 KO mouse exhibited cough and hyposmia phenotype. The loss of Tppp3 disrupted the apical microtubules (MTs) meshwork in the tracheal MCCs, leading to random orientation and alignment of basal bodies (BBs) of the motile cilia. Unexpectedly, aberrant ciliary membrane fusions occurred in the trachea of the Tppp3 KO mice. We examined the underlying molecular mechanism of the ciliary membrane fusion by isolating the tracheal cilium. Liquid Chromatography-Mass Spectrometry (LC–MS) analysis as well as pharmacological analysis revealed that hyperaccumulation of a long chain ceramide at the ciliary membrane caused the membrane fusion. In addition, sensory cilia formation was impaired in the olfactory sensory neuron of the Tppp3 KO mice. Due to the lack of Tppp3, dendritic MT assembly that underlies long-range migration of BBs toward the cell surface was impaired. These findings demonstrate that Tppp3, as well as the defined intracellular MT architecture, regulate proper orientation/subcellular positioning of BBs and the independency of individual motile cilium membranes.

Extracellular vesicles (EVs) mediate the transfer of intracellular proteins from producer to recipient cells. EVs originate either from plasma membrane protrusions or endosomes, with endosome-derived EVs being extensively studied and engineered. However, the efficiency and functionality of protein transfer via both types of EVs remain poorly understood. Here, we demonstrate that natural EVs derived from cell protrusions dependent on the I-BAR protein MIM, rather than from endosomes, deliver the functional small GTPase Rac1 protein at levels similar to microinjection. Rac1-containing EVs are internalized via endocytosis, trafficked through endosomal compartments, and subsequently released into the cytosol, where they enhance cell motility. To evaluate broader applicability, the genome-editing protein Cas12f is packaged into protrusion-derived EVs by MIM and endosome-derived EVs by endosomal tetraspanin CD63. Notably, protrusion-derived EVs deliver Cas12f with significantly higher efficiency than endosome-derived EVs, highlighting their superior capability for functional protein transfer. Our findings establish the protrusion-derived EVs as a powerful platform for the efficient and bioactive delivery of both native and engineered proteins, expanding the EV-based therapeutic strategies.

The kidney proximal tubule (PT) is a specialized polarized epithelium that functions as a high capacity resorptive machine. PT cells are exquisitely sensitive to ischemia due to their high metabolic rate. The small GTPase Rac1 regulates epithelial function by promoting polarity through its effects on the actin cytoskeleton. We show that Rac1, in the setting of the recovery of the PT from ischemic injury, plays a critical role in reconstituting cellular bioenergetics by promoting actin cytoskeleton formation around damaged mitochondria. This mechanism removes damaged mitochondria through mitophagy and preserves PT metabolic capacity and reabsorption function. Loss of Rac1 causes intracellular lipid accumulation, energy depletion, and PT cell atrophy. Thus, Rac1 promotes the repair of PT cells by enhancing mitochondrial bioenergetics, rather than by regulating cell polarity via a mechanism that links the actin cytoskeleton to metabolic demands and cell morphology.

Pore-forming peptides (PFPs) are powerful tools for engineering biological membranes in vitro and in living cells. They can be derived from natural sources or designed de novo , with synthetic peptides offering distinct advantages in rational design unconstrained by natural sequences. While native PFPs have been targeted to specific organelles to modulate their functions, the potential of synthetic PFPs for organelle engineering remains largely unexplored. Here, we show that the 28-amino acid synthetic peptides, SVG28 and its variant SVG28D2, can be selectively targeted to mitochondria for functional alteration. More specifically, molecular dynamics simulations first confirmed the structural stability of the SVG28 nanopore even when fused to the TOM20 transmembrane domain, a mitochondrial targeting sequence. In isolated lipid bilayers, TOM20-SVG28 formed pores with a 0.3-nS, conductance, or 1.0-nm pore. The expression efficiency was further enhanced by two amino acid substitutions (SVG28D2). In cells, both TOM20-SVG28 and TOM20-SVG28D2 localized to the mitochondria, induced morphological alterations, and reduced both membrane potential and ATP production. These observations in silico, in vitro and in cellulo collectively indicate that the synthetic SVG28 peptides self-assemble into pores in the outer mitochondrial membrane, likely permitting proton permeation. Importantly, the matrix protein Su9 remained confined within mitochondria, suggesting preserved inner membrane integrity, and thus specific effect on the target. This work establishes a framework for the design and application of genetically-encoded synthetic PFPs in living cells, opening new avenues for cellular engineering.

Summary: Membrane-less organelles (MLOs) perform diverse cellular functions, but how they interact to coordinate these processes remains poorly understood. Here we constructed a “Rainbow Nucleus” cell line to simultaneously visualize five nuclear MLOs using multi-spectral live-cell imaging. We find that MLO interactions are not random: nuclear speckles serve as hubs, and functionally related MLOs interact more frequently than unrelated MLOs. While some interactions are stable, such as those between the histone locus bodies and Cajal bodies, others are transient, such as those between PML nuclear bodies and Cajal bodies. Single particle tracking revealed that stable contacts between Cajal bodies and nuclear speckles are functional, promoting the outward trafficking of small nuclear ribonucleoproteins from Cajal bodies. Finally, RNA Polymerase II-mediated transcription is required to organize the MLO interactome. Our study reveals a structured, transcription-dependent contact network among nuclear MLOs, laying the foundation for future cellular engineering efforts to modulate the MLO interactome.

Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise and rapid induction of microtubule acetylation within minutes in live cells. Using optoTAT, we observed striking and rapid responses at both molecular and cellular level. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional cell migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation in the regulation of directed cell migration, revealing a dynamic crosstalk between the actin and microtubule cytoskeletal networks.