Biology subjects

Toyama, Y.

Publications and source records attributed to Toyama, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Microdomes: Micro-engineered Microdome arrays enable standardised and shear-free 3D biology with full-spectrum optical imaging compatibility

Three-dimensional cellular models such as organoids and spheroids hold major promise for developmental biology, disease modelling and precision medicine, yet their large-scale production and analysis remain constrained by handling-induced shear stress, sample fragility, positional instability and limited compatibility with advanced imaging workflows. Here, we introduce Microdomes, dense arrays of open-top, dome-shaped microcavities. Each cavity holds a single specimen in its own miniature aquarium, accessed through a narrow apical opening that admits cells, medium, matrix and staining reagents while shielding it from the shear generated during routine pipetting. Each Microdomes chip accommodates more than 100 spheroids or organoids, supporting diverse cell types, co-culture formats and both matrix-free and matrix-embedded culture. All specimens are retained through prolonged culture, repeated medium exchange, fixation and immunostaining. Because every specimen occupies a fixed, addressable position against a thin transparent film, the same spheroid can be relocated and re-imaged over weeks of culture and across microscopes, from array-wide overviews to subcellular details, without transfer, embedding or other perturbation. Microdomes also support AI-based automated segmentation, from whole-spheroid outlines to individual nuclei in 3D, using common image analysis software. Microdomes thereby turn each array into a self-contained quality control unit, providing specimen-level traceability that organoid production pipelines currently lack.

cell biology↗

bioRxiv2026

Chromosome-scale genome assembly of Cycas revoluta provides insights into cycad diversification and species diversity

Cycads are an ancient group of seed plants. Despite their ancient origin, many extant cycad genera exhibit high species diversity. The specialized reproductive traits in cycads, dioecy governed by the XY sex-determination system and the elaborate co-evolutionary synergy with insect pollinators, may facilitate lineage diversification. Here, we present a chromosome-scale genome sequence of Cycas revoluta, the species in which plant spermatozoids were discovered in 1896. The genome sequence spanned 11.6 Gb, 98.4% of which were anchored onto the 11 cycad chromosomes. Repetitive sequences occupied 9.8 Gb, and 31,481 genes were predicted. Based on this genome assembly, the X- and Y-associated genomic regions were characterized, and candidate genes for sex determination were identified. In addition, the genomic positions of genes for sex-related traits were determined. Subsequently, we analyzed transcriptomes for thermogenesis responsible for attracting insect pollinators. Through these comprehensive analyses, we provide new insights into the genomic basis of cycad diversification and species diversity.

genomics↗

bioRxiv2026

A Moonlighting Function of Kinetochore Complex Proteins in Golgi Assembly of Neural Stem Cells

Kinetochores play an essential role in mitosis, and human kinetochore variants are associated with microcephaly. However, their functions in quiescent/interphase cells are unknown. Here, we demonstrate that Drosophila kinetochore proteins promote the reactivation of quiescent NSCs and their regeneration upon injury by organizing Golgi-dependent acentrosomal microtubules. Drosophila kinetochore proteins localize to the Golgi vicinity in quiescent NSCs and are required for proper localization of Golgi proteins, including the Golgi-resident small GTPase Arf1 known to regulate quiescent NSC reactivation and regeneration. Moreover, Drosophila kinetochore proteins physically associate with Arf1 and function upstream of it during reactivation. Remarkably, human kinetochore protein SPC25 localizes to Golgi in interphase NSCs and HEK293T cells. Furthermore, human kinetochores have a conserved function in promoting Golgi organization and, in turn, microtubule dynamics, in interphase cells. Together, our findings reveal a conserved moonlighting role for kinetochores in Golgi assembly in quiescent Drosophila NSCs and human interphase cells.

neuroscience↗

bioRxiv2026

A confining microfluidic platform for disparate density coculture reveals the dynamics of macrophage-mediated adipocyte clearance

Co-culturing cells with mismatched densities, where one cell type adheres to surfaces while the other floats, represents a fundamental challenge in cell biology. This is particularly evident in studying macrophage-adipocyte interactions, where macrophages must engage and clear lipid-rich apoptotic adipocytes, a process critical to understanding chronic inflammation in obesity and metabolic disease. The density disparity between macrophages, which sink and adhere to culture surfaces, and adipocytes, which float due to their lipid content, has prevented conventional co-culture approaches from achieving sustained cell-cell contact. To address this challenge, we developed a microfluidic system that confines adipocytes and lipid droplets in close proximity to macrophages. This platform features recessed micro-traps within the upper surface of a microfluidic chamber that trap buoyant objects while allowing media exchange and delivery of reagents for live-cell and immunofluorescence imaging. Time lapse imaging revealed that the dynamic process of macrophages-dead corpse interactions, showing that individual macrophages cannot engulf entire corpses but instead mechanically deform them. Furthermore, the platform successfully recapitulates the formation of Crown-Like Structures (CLS), clusters of macrophages surrounding dead adipocytes that are hallmarks of adipose tissue inflammation. Long-term culture revealed that CLS effectively clear lipids compared to partial macrophage engagement, providing mechanistic insights that were previously unattainable with standard histological approaches. Beyond the macrophage-lipid interaction, this platform has potential for studying interactions between adherent cells and buoyant targets, such as microplastics, opening new avenues for research where density mismatch poses a major barrier.

bioengineering↗

bioRxiv2026

Elimination of senescent cells by mechanical cell competition

Cellular senescence, a hallmark of aging, leads to the accumulation of apoptosis-resistant cells that compromise tissue homeostasis. While senescent cells are known to influence neighboring cells through the senescence-associated secretory phenotype (SASP), the precise nature of the interactions between senescent and normal cells remains elusive. Here we show that progerin-induced senescent cells undergo apoptosis when co-cultured with normal cells. This elimination requires direct cell-cell contact and is mediated by the JNK and p38-MAPK pathways, leading to p53 upregulation and p21 downregulation in progerin-expressing cells. Furthermore, neighboring normal cells exert persistent mechanical compression on progerin-expressing cells prior to their elimination, consistent with mechanical cell competition. In contrast, p16-induced senescent cells resist elimination under the same co-culture conditions, maintaining high p21 levels. Our findings reveal a non-cell-autonomous mechanism for senescent cell clearance, providing new insights into the maintenance of tissue homeostasis during aging.

cell biology↗

bioRxiv2026

Immune reprogramming of 3D tumor models via optoporation-mediatedtargeted gene delivery to macrophages

The dynamics of the tumor microenvironment (TME) are a key determinant of cancer progression and therapeutic resistance through complex interactions between tumor, stromal and immune cell populations. Among these, tumor-associated macrophages (TAMs) play a central role in promoting tumor growth and immune suppression. However, the specific contributions of TAMs remain poorly understood due to the lack of tools enabling selective genetic manipulation in three-dimensional (3D) tumor models. Here, we present a gold nanoparticle-assisted optoporation approach that enables spatially selective plasmid-based gene delivery to TAMs within intact heterocellular 3D pancreatic ductal adenocarcinoma (PDAC) spheroids, thereby modulating the TME. In two-dimensional (2D) TAM cultures, conventional transfection of IRF5- and IKBKB-encoding plasmids validated their capacity to induce TAM repolarization, as evidenced by activation of interferon signaling. Extending this approach to 3D PDAC spheroids, nanoparticle-assisted optoporation achieved selective transfection of TAMs with IRF5- and IKBKB-encoding plasmids by transiently generating nanoscale membrane pores in illuminated cells. TAMs transfection elicited a robust interferon response, marked by transcriptional upregulation of IFNA, IFNB1, and CXCL10, and increased protein levels of IFNB1, IFNL1, and CXCL13, together with downregulation of pro-tumorigenic markers CEACAM5, IL19, and IL32. These coordinated changes indicate a shift towards an anti-tumorigenic TME. By enabling minimally invasive, TAM-specific gene delivery in complex multicellular 3D spheroids, this strategy allows precise modulation of the TME and opens new avenues for modeling its dynamics in cancer progression and therapeutic response.

bioengineering↗

bioRxiv2025

Intermediate Evolutionary State of Motile Sperm and Pollen Tubes in the Extant Gymnosperm Cycas revoluta

The transformation of male reproductive cells is a significant event in the evolution of land plant fertilization systems from zooidogamy to siphonogamy. Basal plants such as bryophytes and pteridophytes have motile sperm, whereas most seed plants have non-motile sperm, delivered by a pollen tube. Despite being seed plants, gymnosperm cycads and ginkgo uniquely form highly multi-flagellated and large motile sperm within pollen tubes. However, the evolutionary state of these male reproductive cells remains unknown. We clarified the gene expression profiles of Cycas revoluta pollen tubes and motile sperm that swam toward female reproductive cells. Male cycad cells had fewer genes associated with transcription, translation, and related processes, which is consistent across land plants. Similar to other male gametes, a sperm-specific histone variant may contribute to transcriptional regulation via chromatin condensation. Plasma membrane-localized proteins probably involving male-female interactions revealed that cycad sperm express both factors found in motile plant sperm and seed plant sperm cells, suggesting a transitional state from motile to non-motile male gametes. In contrast, cycad pollen tubes lacked plasma membrane-localized receptor genes previously reported in angiosperms and cell wall-associated factors involved in pollen tube tip growth. These results support the hypothesis that the function of the cycad pollen tube is confined to the haustoria as previously reported. These results indicate a molecular intermediate state of the cycad fertilization mechanism with motile sperm and pollen tubes, providing important insights into the evolution of land plant fertilization. Significant StatementCycads and ginkgo, which possess primitive motile sperm and derived pollen tubes, are important for understanding the evolution of land plant fertilization from zooidogamy to siphonogamy. We explored gene expression in living, capacitated motile sperm and pollen tubes of Cycas revoluta in a tissue-specific transcriptome analysis. Similar to other land plants, the transcriptional repression mechanism has been suggested in these male cells. Plasma membrane-localized genes revealed the characteristics of motile and non-motile sperm, while molecular evidence of pollen tube guidance and tip growth, as seen in angiosperms, was lacking in cycads. A molecular intermediate state of the cycad fertilization mechanism was clarified, providing important insights into the evolutionary process of land plant fertilization.

plant biology↗

bioRxiv2024

ARHGAP12 suppresses F-actin assembly to control epithelial tight junction mechanics and paracellular leak pathway permeability

Tight junctions (TJ) separate body compartments and control the paracellular transport of ions, solutes, and macromolecules across epithelial barriers. There is evidence that claudin-based ion transport (the pore pathway) and the paracellular transport of macromolecules (the leak pathway) are independently regulated processes. However, how leak pathway permeability is controlled is not well understood. Here we have identified the Cdc42/Rac GTPase activating protein ARHGAP12 as a novel and specific regulator of the leak pathway. ARHGAP12 is recruited to TJ via an interaction between its SH3 domain and the TJ protein ZO-2. Using a combination of biochemical and biophysical approaches, in vitro actin polymerisation assays, and permeability assays in MDCK-II cells, we show that ARHGAP12 suppresses N-WASP-mediated F-actin assembly at TJ to dampen junctional tension. This promotes paracellular leak pathway permeability without affecting ion flux. Mechanistically, we demonstrate that the ARHGAP12 tandem WW domain interacts directly and in a multivalent manner with an array of PPxR motifs in the proline-rich domain of N-WASP. This interaction is sufficient to suppress SH3 domain-mediated N-WASP oligomerisation and Arp2/3-driven F-actin assembly in vitro. Collectively our data demonstrate a critical role for ARHGAP12 in suppressing junctional F-actin assembly and tension to promote the flux of small macromolecules across the TJ.

cell biology↗

bioRxiv2024

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein q subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

developmental biology↗

bioRxiv2022

Golgi-dependent Reactivation and Regeneration of Quiescent Neural Stem Cells

The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. In Drosophila, quiescent neural stem cells (qNSCs) extend a primary protrusion, which is removed prior to NSC reactivation. Here, we have unravelled that qNSC protrusions can be regenerated upon injury. This regeneration process relies on the Golgi apparatus which acts as the major acentrosomal microtubule-organizing centre in qNSCs. A Golgi-resident GTPase Arf1 and its guanine-nucleotide exchange factor Sec71 promote NSC reactivation and regeneration via the regulation of microtubule growth. Arf1 physically associates with its new effector Mini Spindles (Msps)/XMAP215, a microtubule polymerase. Finally, Arf1 functions upstream of Msps to target the cell-adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. Our findings have established Drosophila qNSCs as a new regeneration model and identified a novel Arf1/Sec71-Msps pathway in the regulation of microtubule growth and NSC reactivation.

cell biology↗

bioRxiv2022

Mechanical stress driven by rigidity sensing governs epithelial stability

Epithelia act as a barrier against environmental stress and abrasion and in vivo they are continuously exposed to environments of various mechanical properties. The impact of this environment on epithelial integrity remains elusive. By culturing epithelial cells on 2D hydrogels, we observe a loss of epithelial monolayer integrity through spontaneous hole formation when grown on soft substrates. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from tensile on soft to compressive on stiff substrates. Through active nematic modelling, we find unique patterns of cell shape texture called nematic topological defects that underpin large isotropic stress fluctuations at certain locations thereby triggering mechanical failure of the monolayer and hole opening. Our results show that substrate stiffness provides feedback on monolayer mechanical state and that topological defects can trigger stochastic mechanical failure, with potential application towards a mechanistic understanding of compromised epithelial integrity in bacterial infection, tumor progression and morphogenesis.

biophysics↗

bioRxiv2021

Mechanics defines the spatial pattern of compensatory proliferation

The number of cells in tissues is tightly controlled by cell division and cell death, and misregulation of cell numbers could lead to pathological conditions such as cancer. To maintain cell numbers in a tissue, a cell elimination process named programmed cell death or apoptosis, stimulates the proliferation of neighboring cells. This mechanism is called apoptosis-induced compensatory proliferation, which was originally reported more than 40 years ago. While only a limited number of the neigboring cells need to divide to compensate for apoptotic cell loss, the mechanisms that select cells for undergoing division remain an open question. Here we found that the spatial inhomogeneity in mechanotransduction through a growth-promoting transcription co-activator Yes-associated protein (YAP) in the neighboring tissue, accounts for the inhomogeneity of compensatory proliferation. Such inhomogeneous mechanotransduction arises from the combination of the non-uniform distribution of nuclear size, which is inherent in tissues, and the non-uniform pattern of mechanical force applied to the neighboring cells upon apoptosis. Our findings from a mechanical perspective complement the current biochemical understanding of compensatory growth and provide additional insights into cellular functions of how tissue precisely maintains its homeostasis.

cell biology↗

bioRxiv2021

CZON-cutter: a CRISPR-Cas9 system with multiplexed organelle imaging in a simple unicellular alga

The simple cellular structure of the unicellular alga Cyanidioschyzon merolae consists of one nucleus, one mitochondrion, one chloroplast, and one peroxisome per cell and offers unique advantages to investigate mechanisms of organellar proliferation and the cell cycle. Here, we describe an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, CZON-cutter, for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease to target editing at a locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants by fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows validation of phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. SummaryAn engineered CRISPR-Cas9 system, named CZON-cutter, for simultaneous genome editing and fluorescent protein labeling of organelles in Cyanidioschyzon merolae can be used to validate intracellular function of a particular gene, even if it is essential.

molecular biology↗

bioRxiv2021

Gene expression shapes the patterns of parallel evolution of herbicide resistance in the agricultural weed Monochoria vaginalis

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterisation and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

plant biology↗

bioRxiv2020

Nature of active forces in tissues: how contractile cells can form extensile monolayers

Actomyosin machinery endows cells with contractility at a single cell level. However, at a tissue scale, cells can show either contractile or extensile behaviour based on the direction of pushing or pulling forces due to neighbour interactions or substrate interactions. Previous studies have shown that a monolayer of fibroblasts behaves as a contractile system1 while a monolayer of epithelial cells2,3 or neural crest cells behaves as an extensile system.4 How these two contradictory sources of force generation can coexist has remained unexplained. Through a combination of experiments using MDCK (Madin Darby Canine Kidney) cells, and in-silico modeling, we uncover the mechanism behind this switch in behaviour of epithelial cell monolayers from extensile to contractile as the weakening of intercellular contacts. We find that this switch in active behaviour also promotes the buildup of tension at the cell-substrate interface through an increase in actin stress fibers and higher traction forces. This in turn triggers a mechanotransductive response in vinculin translocation to focal adhesion sites and YAP (Yes-associated protein) transcription factor activation. Our studies also show that differences in extensility and contractility act to sort cells, thus determining a general mechanism for mechanobiological pattern formation during cell competition, morphogenesis and cancer progression.

biophysics↗

bioRxiv2020

Adhesion-mediated heterogeneous actin organization governs apoptotic cell extrusion

Apoptotic extrusion is crucial in maintaining epithelial homeostasis and has implications in diseases of epithelial tissues. Current literature supports that epithelia respond to extrusion to maintain their integrity by the formation of a supracellular actomyosin ring (purse-string) in the neighbors that encompasses the dying cells. However, little is known about whether other types of actin structures could contribute to extrusion as well as how forces generated by mechanosensitive proteins in the cells are integrated. Here, we found that during extrusion, a heterogeneous actin network composed of lamellipodia protrusions and discontinuous actomyosin cables, was reorganized in the neighboring cells and was the main factor driving extrusion forwards. The early presence of basal lamellipodia protrusion participated both in basal sealing of the extrusion site and in orienting the actomyosin purse-string at the later stage of extrusion. These sequential events are essential in ensuring a successful extrusion in apicobasal direction. The co-existence of these two mechanisms is determined by the interplay between the cell-cell and cell-substrate adhesions. A theoretical model integrates the role of these cellular mechanosensitive components to explain why a dual-mode mechanism, which combined lamellipodia protrusion and purse-string contractility, leads to more efficient extrusion than a single-mode mechanism. We anticipate that our approach will be useful to provide mechanistic insight into epithelial homeostasis, morphogenetic events and tumorigenesis.

cell biology↗

bioRxiv2020

Leaf trichome distribution pattern in Arabidopsis reveals gene expression variation associated with environmental adaptation

Gene expression varies stochastically even in both heterogenous and homogeneous cell populations. This variation is not simply useless noise; rather, it is important for many biological processes. Unicellular organisms or cultured cell lines are useful for analyzing the variation in gene expression between cells; however, owing to technical challenges, the biological relevance of this variation in multicellular organisms such as higher plants remains unclear. Here, we addressed the biological relevance of this variation between cells by examining the genetic basis of trichome distribution patterns in Arabidopsis thaliana. The distribution pattern of a trichome on a leaf is stochastic and can be mathematically represented using Turings reaction-diffusion (RD) model. We analyzed simulations based on the RD model and found that the variability in the trichome distribution pattern increased with the increase in stochastic variation in a particular gene expression. Moreover, differences in heat-dependent variability of the trichome distribution pattern between the accessions showed a strong correlation with environmental factors to which each accession was adapted. Taken together, we successfully visualized variations in gene expression by quantifying the variability in the Arabidopsis trichome distribution pattern. Thus, our data provide evidence for the biological importance of variations in gene expression for environmental adaptation.

plant biology↗

bioRxiv2019

Desmosomal coupling in apoptotic cell extrusion

The mechanical coupling of epithelia enables coordination of tissue functions and collective tissue movements during different developmental and physiological processes. This coupling is ensured by cell-cell junctions, including adherens junctions (AJs) and desmosomal junctions (DJs) [1, 2]. During apoptosis, or programmed cell death, a dead cell is expelled from the tissue by coordinated processes between the dying cell and its neighbors. Apoptotic cell extrusion is driven by actomyosin cable formation and its contraction, and lamellipodial crawling of the neighboring cells (Fig. S1A-A, Movie S1) [3-6]. Throughout cell extrusion, the mechanical coupling of epithelia needs to be maintained in order to preserve tissue homeostasis [3]. Although much is known about the regulation of AJs in apoptotic cell extrusion [6-9], the role and dynamics of DJs during this process remains poorly understood. Here, we show that DJs stay intact throughout and are crucial for apoptotic cell extrusion. Pre-existing DJs between the apoptotic cell and neighboring non-dying cells remain intact even during the formation of de novo DJs between non-dying cells, suggesting that the neighboring cells possess two DJs in the middle of apoptotic cell extrusion. We further found that an actomyosin cable formed in the vicinity of DJs upon apoptosis, and subsequently deviated from DJs during its constriction. Interestingly, the departure of the actomyosin cable from DJs coincided with the timing when DJs lost their straightness, suggesting a release of junctional tension at DJs, and a mechanical coupling between DJs and actomyosin contractility. The depletion of desmoplakin, which links desmosomes and intermediate filaments, resulted in defective apical contraction and an inability to form de novo DJs, leading to a failure of apoptotic cell extrusion. Our study provides a framework to explain how desmosomes play pivotal roles in maintaining epithelial sheet integrity during apoptotic cell extrusion.\n\nO_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC=\"FIGDIR/small/816280v1_figs1.gif\" ALT=\"Figure 1\">\nView larger version (49K):\norg.highwire.dtl.DTLVardef@454179org.highwire.dtl.DTLVardef@cd7e75org.highwire.dtl.DTLVardef@1295ed1org.highwire.dtl.DTLVardef@1102c8f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure S1:C_FLOATNO Desmosomal junctions at the interface during apoptotic extrusion. A) Confocal images showing actin (Lifeact-Ruby) dynamics during apoptotic cell extrusion in both apical (top panel) and basal (bottom panel) sections of the neighboring cell. Blue-filled and open arrows show the actomyosin cable (top panel) and the lamellipodia (bottom panel) respectively. A) Illustration showing the definition of interface ROI during extrusion. A) Graphs plotting normalized apoptotic cell area (black) and actin (blue) intensity at the dying cell interface during extrusion (n=5 independent experiments). B) Confocal images showing the localization of overexpressed Dsg-FLAG-EGFP (green) and endogenous desmoplakin (red) in MDCK monolayers (n=3 independent staining experiments). B) Magnified images of the white-filled and dotted boxes from B showing co-existence of dotted and continuous junctional clusters in the cell. C) Confocal images showing mCherry-E-cadherin dynamics at the interface during extrusion. C) Graphs plotting normalized apoptotic cell area (black) and E-Cadherin (blue) intensity during constriction (n=5 independent experiments). D) Confocal images showing desmoglein 2 and membrane (mCherry-delta-PH) dynamics in both apical (top panel) and basal (bottom panel) sections of the cell. White arrows (bottom panel) highlights formation of de novo junctions (n=8 independent experiments). E) Confocal images of endogenous desmosomes in the apical (top panel) and basal (bottom panel) sections during different phases of cell extrusion. Yellow and white arrows indicate pre-existing and de novo junctions, respectively (n=182 extrusion events from 7 independent experiments). F) Confocal images showing desmosome (green), killer red (mem-Killer red, cyan) and caspase 3 (magenta) before and after photo-irradiation. White arrow denotes extent of the dying cell. F) Confocal images showing desmosome dynamics (black) in the apical (top panel) and basal (bottom panel) sections of the neighboring cell. Red and blue arrows highlight pre-existing and de novo junctions respectively. F) Transverse view of a neighboring cell showing DJ dynamics after apoptosis induction through Killer Red. Red and blue arrows indicate pre-existing and de novo junctions respectively (n=4 independent experiments). The data from A and C represent mean {+/-}SEM. Scale bars, 10 m except C (5 m).\n\nC_FIG

cell biology↗