Linking phosphoinositide function to mitosis.

Phosphoinositides (PtdIns) are a family of differentially phosphorylated lipid second messengers localized to the cytoplasmic leaflet of both plasma and intracellular membranes. Kinases and phosphatases can selectively modify the PtdIns composition of different cellular compartments, leading to the recruitment of specific binding proteins, which control cellular homeostasis and proliferation. Thus, while PtdIns affect cell growth and survival during interphase, they are also emerging as key drivers in multiple temporally defined membrane remodeling events of mitosis, like cell rounding, spindle orientation, cytokinesis, and abscission. In this review, we summarize and discuss what is known about PtdIns function during mitosis and how alterations in the production and removal of PtdIns can interfere with proper cell division.

VAP-mediated membrane-tethering mechanisms implicate ER-PM contact function in pH homeostasis.

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are highly conserved endoplasmic reticulum (ER)-resident proteins that establish ER contacts with multiple membrane compartments in many eukaryotes. However, VAP-mediated membrane-tethering mechanisms remain ambiguous. Here, focusing on fission yeast ER-plasma membrane (PM) contact formation, using systematic interactome analyses and quantitative microscopy, we predict a non-VAP-protein direct binding-based ER-PM coupling. We further reveal that VAP-anionic phospholipid interactions may underlie ER-PM association and define the pH-responsive nature of VAP-tethered membrane contacts. Such conserved interactions with anionic phospholipids are generally defective in amyotrophic lateral sclerosis-associated human VAPB mutant. Moreover, we identify a conserved FFAT-like motif locating at the autoinhibitory hotspot of the essential PM proton pump Pma1. This modulatory VAP-Pma1 interaction appears crucial for pH homeostasis. We thus propose an ingenious strategy for maintaining intracellular pH by coupling Pma1 modulation with pH-sensory ER-PM contacts via VAP-mediated interactions.

Extracellular-vesicle-packaged S100A11 from osteosarcoma cells mediates lung premetastatic niche formation by recruiting gMDSCs.

The premetastatic niche (PMN) contributes to lung-specific metastatic tropism in osteosarcoma. However, the crosstalk between primary tumor cells and lung stromal cells is not clearly defined. Here, we dissect the composition of immune cells in the lung PMN and identify granulocytic myeloid-derived suppressor cell (gMDSC) infiltration as positively associated with immunosuppressive PMN formation and tumor cell colonization. Osteosarcoma-cell-derived extracellular vesicles (EVs) activate lung interstitial macrophages to initiate the influx of gMDSCs via secretion of the chemokine CXCL2. Proteomic profiling of EVs reveals that EV-packaged S100A11 stimulates the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway in macrophages by interacting with USP9X. High level of S100A11 expression or circulating gMDSCs correlates with the presentation of lung metastasis and poor prognosis in osteosarcoma patients. In summary, we identify a key role of tumor-derived EVs in lung PMN formation, providing potential strategies for monitoring or preventing lung metastasis in osteosarcoma.

Origin of eukaryotic-like Vps23 shapes an ancient functional interplay between ESCRT and ubiquitin system in Asgard archaea.

Functional interplay between the endosomal sorting complexes required for transport (ESCRT) and the ubiquitin system underlies the ubiquitin-dependent cargo-sorting pathway of the eukaryotic endomembrane system, yet its evolutionary origin remains unclear. Here, we show that a UEV-Vps23 protein family, which contains UEV and Vps23 domains, mediates an ancient ESCRT and ubiquitin system interplay in Asgard archaea. The UEV binds ubiquitin with high affinity, making the UEV-Vps23 a sensor for sorting ubiquitinated cargo. A steadiness box in the Vps23 domain undergoes ubiquitination through an Asgard E1, E2, and RING E3 cascade. The UEV-Vps23 switches between autoinhibited and active forms, regulating the ESCRT and ubiquitin system interplay. Furthermore, the shared sequence and structural homology among the UEV-Vps23, eukaryotic Vps23, and archaeal CdvA suggest a common evolutionary origin. Together, this work expands our understanding of the ancient ESCRT and ubiquitin system interplay that likely arose antedating divergent evolution between Asgard archaea and eukaryotes.

Insect-transmitted plant virus balances its vertical transmission through regulating Rab1-mediated receptor localization.

Rice stripe virus (RSV) establishes infection in the ovaries of its vector insect, Laodelphax striatellus. We demonstrate that RSV infection delays ovarian maturation by inhibiting membrane localization of the vitellogenin receptor (VgR), thereby reducing the vitellogenin (Vg) accumulation essential for egg development. We identify the host protein L. striatellus Rab1 protein (LsRab1), which directly interacts with RSV nucleocapsid protein (NP) within nurse cells. LsRab1 is required for VgR surface localization and ovarian Vg accumulation. RSV inhibits LsRab1 function through two mechanisms: NP binding LsRab1 prevents GTP binding, and NP binding LsRab1-GTP complexes stimulates GTP hydrolysis, forming an inactive LsRab1 form. Through this dual inhibition, RSV infection prevents LsRab1 from facilitating VgR trafficking to the cell membrane, leading to inefficient Vg uptake. The Vg-VgR pathway is present in most oviparous animals, and the mechanisms detailed here provide insights into the vertical transmission of other insect-transmitted viruses of medical and agricultural importance.

"Forcing" new interpretations of molecular tension sensor studies.

Molecular tension sensors are central tools for mechanobiology studies but have limitations in interpretation. Reporting in Cell Reports Methods, Shoyer et al. discover that fluorescent protein photoswitching in concert with sensor extension may expand the use and interpretation of common force-sensing tools.

Arginine methylation-enabled FUS phase separation with SMN contributes to neuronal granule formation.

Various ribonucleoprotein complexes (RNPs) often function in the form of membraneless organelles derived from multivalence-driven liquid-liquid phase separation (LLPS). Post-translational modifications, such as phosphorylation and arginine methylation, govern the assembly and disassembly of membraneless organelles. This study reveals that asymmetric dimethylation of arginine can create extra binding sites for multivalent Tudor domain-containing proteins like survival of motor neuron (SMN) protein, thereby lowering the threshold for LLPS of RNPs, such as fused in sarcoma (FUS). Accordingly, FUS hypomethylation or knockdown of SMN disrupts the formation and transport of neuronal granules in axons. Wild-type SMN, but not the spinal muscular atrophy-associated form of SMN, SMN-Δ7, rescues neuronal defects due to SMN knockdown. Importantly, a fusion of SMN-Δ7 to an exogenous oligomeric protein is sufficient to rescue axon length defects caused by SMN knockdown. Our findings highlight the significant role of arginine methylation-enabled multivalent interactions in LLPS and suggest their potential impact on various aspects of neuronal activities in neurodegenerative diseases.

Tumor suppressor KEAP1 promotes HSPA9 degradation, controlling mitochondrial biogenesis in breast cancer.

The oxidative-stress-related protein Kelch-like ECH-associated protein 1 (KEAP1) is a substrate articulator of E3 ubiquitin ligase, which plays an important role in the ubiquitination modification of proteins. However, the function of KEAP1 in breast cancer and its impact on the survival of patients with breast cancer remain unclear. Our study demonstrates that KEAP1, a positive prognostic factor, plays a crucial role in regulating cell proliferation, apoptosis, and cell cycle transition in breast cancer. We investigate the underlying mechanism using human tumor tissues, high-throughput detection technology, and a mouse xenograft tumor model. KEAP1 serves as a key regulator of cellular metabolism, the reprogramming of which is one of the hallmarks of tumorigenesis. KEAP1 has a significant effect on mitochondrial biogenesis and oxidative phosphorylation by regulating HSPA9 ubiquitination and degradation. These results suggest that KEAP1 could serve as a potential biomarker and therapeutic target in the treatment of breast cancer.

Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.

Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca2+, across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites.

SRRM2 phase separation drives assembly of nuclear speckle subcompartments.

Nuclear speckles (NSs) are nuclear biomolecular condensates that are postulated to form by macromolecular phase separation, although the detailed underlying forces driving NS formation remain elusive. SRRM2 and SON are 2 non-redundant scaffold proteins for NSs. How each individual protein governs assembly of the NS protein network and the functional relationship between SRRM2 and SON are largely unknown. Here, we uncover immiscible multiphases of SRRM2 and SON within NSs. SRRM2 and SON are functionally independent, specifically regulating alternative splicing of subsets of mRNA targets, respectively. We further show that SRRM2 forms multicomponent liquid phases in cells to drive NS subcompartmentalization, which is reliant on homotypic interaction and heterotypic non-selective protein-RNA complex coacervation-driven phase separation. SRRM2 serine/arginine-rich (RS) domains form higher-order oligomers and can be replaced by oligomerizable synthetic modules. The serine residues within the RS domains, however, play an irreplaceable role in fine-tuning the liquidity of NSs.

The Hippo signaling pathway in development and regeneration.

The Hippo signaling pathway is a central growth control mechanism in multicellular organisms. By integrating diverse mechanical, biochemical, and stress cues, the Hippo pathway orchestrates proliferation, survival, differentiation, and mechanics of cells, which in turn regulate organ development, homeostasis, and regeneration. A deep understanding of the regulation and function of the Hippo pathway therefore holds great promise for developing novel therapeutics in regenerative medicine. Here, we provide updates on the molecular organization of the mammalian Hippo signaling network, review the regulatory signals and functional outputs of the pathway, and discuss the roles of Hippo signaling in development and regeneration.

TRABD modulates mitochondrial homeostasis and tissue integrity.

High TRABD expression is associated with tau pathology in patients with Alzheimer's disease; however, the function of TRABD is unknown. Human TRABD encodes a mitochondrial outer-membrane protein. The loss of TRABD resulted in mitochondrial fragmentation, and TRABD overexpression led to mitochondrial clustering and fusion. The C-terminal tail of the TRABD anchored to the mitochondrial outer membrane and the TraB domain could form homocomplexes. Additionally, TRABD forms complexes with MFN2, MIGA2, and PLD6 to facilitate mitochondrial fusion. Flies lacking dTRABD are viable and have normal lifespans. However, aging flies exhibit reduced climbing ability and abnormal mitochondrial morphology in their muscles. The expression of dTRABD is increased in aged flies. dTRABD overexpression leads to neurodegeneration and enhances tau toxicity in fly eyes. The overexpression of dTRABD also increased reactive oxygen species (ROS), ATP production, and protein turnover in the mitochondria. This study suggested that TRABD-induced mitochondrial malfunctions contribute to age-related neurodegeneration.

In vivo imaging in transgenic songbirds reveals superdiffusive neuron migration in the adult brain.

Neuron migration is a key phase of neurogenesis, critical for the assembly and function of neuronal circuits. In songbirds, this process continues throughout life, but how these newborn neurons disperse through the adult brain is unclear. We address this question using in vivo two-photon imaging in transgenic zebra finches that express GFP in young neurons and other cell types. In juvenile and adult birds, migratory cells are present at a high density, travel in all directions, and make frequent course changes. Notably, these dynamic migration patterns are well fit by a superdiffusive model. Simulations reveal that these superdiffusive dynamics are sufficient to disperse new neurons throughout the song nucleus HVC. These results suggest that superdiffusive migration may underlie the formation and maintenance of nuclear brain structures in the postnatal brain and indicate that transgenic songbirds are a useful resource for future studies into the mechanisms of adult neurogenesis.

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-µm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.

Isoform-specific sequestration of protein kinase A fine-tunes intracellular signaling during heat stress.

Protein kinase A (PKA) is a conserved kinase crucial for fundamental biological processes linked to growth, development, and metabolism. The PKA catalytic subunit is expressed as multiple isoforms in diverse eukaryotes; however, their contribution to ensuring signaling specificity in response to environmental cues remains poorly defined. Catalytic subunit activity is classically moderated via interaction with an inhibitory regulatory subunit. Here, a quantitative mass spectrometry approach is used to examine heat-stress-induced changes in the binding of yeast Tpk1-3 catalytic subunits to the Bcy1 regulatory subunit. We show that Tpk3 is not regulated by Bcy1 binding but, instead, is deactivated upon heat stress via reversible sequestration into cytoplasmic granules. These "Tpk3 granules" are enriched for multiple PKA substrates involved in various metabolic processes, with the Hsp42 sequestrase required for their formation. Hence, regulated sequestration of Tpk3 provides a mechanism to control isoform-specific kinase signaling activity during stress conditions.

Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Zn2+-dependent functional switching of ERp18, an ER-resident thioredoxin-like protein.

ERp18 is an endoplasmic reticulum (ER)-resident thioredoxin (Trx) family protein, similar to cytosolic Trx1. The Trx-like domain occupies a major portion of the whole ERp18 structure, which is postulated to be an ER paralog of cytosolic Trx1. Here, we elucidate that zinc ion (Zn2+) binds ERp18 through its catalytic motif, triggering oligomerization of ERp18 from a monomer to a trimer. While the monomeric ERp18 has disulfide oxidoreductase activity, the trimeric ERp18 acquires scavenger activity for hydrogen peroxide (H2O2) in the ER. Depletion of ERp18 thus causes the accumulation of H2O2, which is produced during the oxidative folding of nascent polypeptides in the ER. ERp18 knockdown in C. elegans without Prx4 and GPx7/8, both of which are also known to have H2O2 scavenging activity in the ER, shortened the lifespan, suggesting that ERp18 may form a primitive and essential H2O2 scavenging system for the maintenance of redox homeostasis in the ER.

E-cadherin biomaterials reprogram collective cell migration and cell cycling by forcing homeostatic conditions.

Cells attach to the world through either cell-extracellular matrix adhesion or cell-cell adhesion, and traditional biomaterials imitate the matrix for integrin-based adhesion. However, materials incorporating cadherin proteins that mimic cell-cell adhesion offer an alternative to program cell behavior and integrate into living tissues. We investigated how cadherin substrates affect collective cell migration and cell cycling in epithelia. Our approach involved biomaterials with matrix proteins on one-half and E-cadherin proteins on the other, forming a "Janus" interface across which we grew a single sheet of cells. Tissue regions over the matrix side exhibited normal collective dynamics, but an abrupt behavior shift occurred across the Janus boundary onto the E-cadherin side, where cells attached to the substrate via E-cadherin adhesions, resulting in stalled migration and slowing of the cell cycle. E-cadherin surfaces disrupted long-range mechanical coordination and nearly doubled the length of the G0/G1 phase of the cell cycle, linked to the lack of integrin focal adhesions on the E-cadherin surface.

Temperature change elicits lipidome adaptation in the simple organisms Mycoplasma mycoides and JCVI-syn3B.

Cell membranes mediate interactions between life and its environment, with lipids determining their properties. Understanding how cells adjust their lipidomes to tune membrane properties is crucial yet poorly defined due to the complexity of most organisms. We used quantitative shotgun lipidomics to study temperature adaptation in the simple organism Mycoplasma mycoides and the minimal cell JCVI-syn3B. We show that lipid abundances follow a universal logarithmic distribution across eukaryotes and bacteria, with comparable degrees of lipid remodeling for adaptation regardless of lipidomic or organismal complexity. Lipid features analysis demonstrates head-group-specific acyl chain remodeling as characteristic of lipidome adaptation; its deficiency in Syn3B is associated with impaired homeoviscous adaptation. Temporal analysis reveals a two-stage cold adaptation process: swift cholesterol and cardiolipin shifts followed by gradual acyl chain modifications. This work provides an in-depth analysis of lipidome adaptation in minimal cells, laying a foundation to probe the design principles of living membranes.

Rho-associated kinase regulates Langerhans cell morphology and responsiveness to tissue damage.

Skin damage requires efficient immune cell responses to restore organ function. Epidermal-resident immune cells known as Langerhans cells use dendritic protrusions to surveil the skin microenvironment, which contains keratinocytes and peripheral axons. The mechanisms governing Langerhans cell dendrite dynamics and responses to tissue damage are poorly understood. Using skin explants from adult zebrafish, we show that Langerhans cells maintain normal surveillance following axonal degeneration and use their dendrites to engulf small axonal debris. By contrast, a ramified-to-rounded shape transition accommodates the engulfment of larger keratinocyte debris. We find that Langerhans cell dendrites are populated with actin and sensitive to a broad-spectrum actin inhibitor. We show that Rho-associated kinase (ROCK) inhibition leads to elongated dendrites, perturbed clearance of large debris, and reduced Langerhans cell migration to epidermal wounds. Our work describes the dynamics of Langerhans cells and involvement of the ROCK pathway in immune cell responses.