Biology subjects

Fujimori, T.

Publications and source records attributed to Fujimori, T..

28 records · Page 2Linked to original sources

bioRxiv2022

Differential cellular stiffness contributes to tissue elongation on an expanding surface

Pattern formation and morphogenesis of cell populations is essential for successful embryogenesis. Steinberg proposed the differential adhesion hypothesis (DAH), and differences in cell-cell adhesion and interfacial tension have proven to be critical for cell sorting. Standard theoretical models such as the vertex model consider not only cell-cell adhesion/tension but also area elasticity of apical cell surfaces and viscous friction forces. However, the potential contributions of the latter two parameters to pattern formation and morphogenesis remain to be determined. In this theoretical study, we analyzed the effect of both area elasticity and the coefficient of friction on pattern formation and morphogenesis. We assumed the presence of two cell populations, one population of which is surrounded by the other. Both populations were placed on the surface of a uniformly expanding environment analogous to growing embryos, in which friction forces are exerted between cell populations and their expanding environment. When the area elasticity or friction coefficient in the cell cluster was increased relative to that of the surrounding cell population, the cell cluster was elongated. In comparison with experimental observations, elongation of the notochord in mice is consistent with the hypothesis based on the difference in area elasticity but not the difference in friction coefficient. Because area elasticity is an index of cellular stiffness, we propose that differential cellular stiffness may contribute to tissue elongation within an expanding environment.

biophysics↗

bioRxiv2021

Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells

In mammalian cells genes that are in close proximity are coupled transcriptionally: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also lead to downstream gene silencing, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. We propose a new model of multi-gene regulation, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment.

synthetic biology↗

bioRxiv2021

Tracheal motile cilia require CAMSAP3 for formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a transition zone. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.

cell biology↗

bioRxiv2020

Force-dependent remodeling of a tight junction protein ZO-1 is regulated by phase separation

Although the physiological importance of biomolecular condensates is widely recognized, how it is controlled in time and space during development is largely unknown. Here we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows and internal pressure on TE cells increases. In contrast, this dissolution was less evident in TE cells attached to the inner cell mass, as they receive weaker tensile forces. Furthermore, analyses using MDCK cells have demonstrated that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via an interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to establishing robust cell-cell adhesion during early development.

cell biology↗

bioRxiv2020

Nanobody-mediated control of gene expression and epigenetic memory

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we developed a new strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

synthetic biology↗

bioRxiv2020

Micro-topographical guidance of macropinocytic signaling patches

In fast moving cells such as amoeba and immune cells, spatio-temporal regulation of dendritic actin filaments shapes large-scale plasma membrane protrusions. Despite the importance in migration as well as in particle and liquid ingestion, how these processes are affected by the micrometer-scale surface features is poorly understood. Here, through quantitative imaging analysis of Dictyostelium on micro-fabricated surfaces, we show that there is a distinct mode of topographically guided cell migration phagotaxis directed by the macropinocytic Ras/PI3K signaling patches. The topography guidance was PI3K-dependent and involved nucleation of a patch at the convex curved surface and confinement at the concave surface. Due to the topography-dependence, constitutive cup formation for liquid uptake in the axenic strain is also destined to trace large surface features. Given the fact that PI3K-dependency of phagocytosis are restricted to large particles in both Dictyostelium and immune cells, topography-dependency and the dual-use of membrane cups may be wide-spread.

biophysics↗

bioRxiv2020

Celsr1 and CAMSAP3 differently regulate intercellular and intracellular cilia orientation in oviduct multiciliated cells

The molecular mechanisms by which cilia orientation is coordinated within and between multiciliated cells (MCCs) is not fully understood. By observing the orientation of basal bodies (BB) in MCCs of mouse oviducts, here, we show that Celsr1, a planar cell polarity (PCP) factor involved in tissue polarity regulation, is dispensable for determining BB orientation in individual cells, whereas CAMSAP3, a microtubule minus-end regulator, is critical for this process but not for PCP. MCCs exhibit a characteristic BB orientation and microtubule gradient along the tissue axis, and these intracellular polarities were maintained in the cells lacking Celsr1, although the intercellular coordination of the polarities was partly disrupted. On the other hand, CAMSAP3 regulated the assembly of microtubules interconnecting BBs by localizing at the BBs, and its mutation led to disruption of intracellular coordination of BB orientation, but not affecting PCP factor localization. Thus, both Celsr1 and CAMSAP3 are responsible for BB orientation but in distinct ways; and therefore, their cooperation should be critical for generating functional multiciliated tissues.

cell biology↗

bioRxiv2019

Effective physical properties of cell-cell interaction explain basic structural units of three-dimensional morphogenesis

Mechanical forces are critical for the emergence of diverse three-dimensional morphologies of multicellular systems. However, it remains unclear what kind of mechanical parameters at cellular level substantially contribute to tissue morphologies. This is largely due to technical limitations of live measurements of cellular forces. Here we developed a framework for inferring and modeling mechanical forces of cell-cell interactions. First, by analogy to coarse-grained models in molecular and colloidal sciences, we approximated cells as particles, where mean forces (i.e. effective forces) of pairwise cell-cell interactions are considered. Then, the forces were statistically inferred by fitting the mathematical model to cell tracking data. This method was validated by using synthetic cell tracking data resembling various in vivo situations. Application of our method to the cells in the early embryos of mice and the nematode Caenorhabditis elegans revealed that cell-cell interaction forces can be written as a pairwise potential energy in a manner dependent on cell-cell distances. Importantly, the profiles of the pairwise potentials were quantitatively different among species and embryonic stages, and the quantitative differences correctly described the differences of their morphological features such as spherical vs. distorted cell aggregates, and tightly vs. non-tightly assembled aggregates. We conclude that the effective pairwise potential of cell-cell interactions is a live measurable parameter whose quantitative differences can be a parameter describing three-dimensional tissue morphologies. Author summaryEmergence of diverse three-dimensional morphologies of multicellular organisms is one of the most intriguing phenomena in nature. Due to the complex situations in living systems (e.g. a lot of genes are involved in morphogenesis.), a model for describing the emergent properties of multicellular systems has not been established. To approach this issue, approximation of the complex situations to limited numbers of parameters is required. Here, we searched for mechanical parameters for describing morphologies. We developed a statistical method for inferring mechanical potential energy of cell-cell interactions in three-dimensional tissues; the mechanical potential is an approximation of various mechanical components such as cell-cell adhesive forces, cell surface tensions, etc. Then, we showed that the quantitative differences in the potential is sufficient to reproduce basic three-dimensional morphologies observed during the mouse and C. elegans early embryogenesis, revealing a direct link between cellular level mechanical parameters and three-dimensional morphologies. Our framework provides a noninvasive tool for measuring spatiotemporal cellular forces, which would be useful for studying morphogenesis of larger tissues including organs and their regenerative therapy.

biophysics↗

bioRxiv2019

Isotropic expansion of external environment induces tissue elongation and collective cell alignment

Cell movement is crucial for morphogenesis in multicellular organisms. Growing embryos or tissues often expand isotropically, i.e., uniformly, in all dimensions. On the surfaces of these expanding environments, which we call \"fields,\" cells are subjected to frictional forces and move passively in response. However, the potential roles of isotropically expanding fields in morphogenetic events have not been investigated well. In this study, we mathematically analyzed the effect of isotropically expanding fields using a vertex model, a standard type of multi-cellular model. We found that cells located on fields were elongated along a similar direction each other. Simultaneously, the cell clusters were also elongated, even though field expansion was absolutely isotropic. We then investigated the mechanism underlying these counterintuitive phenomena. In particular, we asked whether elongation was caused by the properties of the field, the cell cluster, or both. Theoretical analyses involving simplification of the model revealed that cell clusters have an intrinsic ability to asymmetrically deform, leading to their elongation. Importantly, this ability is effective only under the non-equilibrium conditions provided by field expansion. This may explain the elongation of the notochord, located on the surface of the growing mouse embryo. We established that passive cell movement induced by isotropically expanding external environments can contribute to both cell and tissue elongation, as well as collective cell alignment, providing key insight into morphogenesis involving multiple adjacent tissues.\n\nStatement of SignificanceIt is a central question of developmental biology how the symmetric shapes of eggs can develop the asymmetric structures of embryos. Embryos expand through their growth. Simultaneously, elongation of tissues such as the notochord occurs, which is fundamental phenomena of morphogenesis. However, possible relationships between tissue elongation and the expansion of embryos have not been investigated well. Here we mathematically present that, even if the expansion is isotropic, tissues located on the embryos are asymmetrically deformed by the expansion, resulting in elongation. We generalize the effect of expanding environments on tissue elongation through model reduction and uncover the mechanism underlying elongation. This process can be a novel key piece for symmetry breaking of embryos, together with previously established morphogenetic processes.

biophysics↗

bioRxiv2018

Tissue self-organization based on collective cell migration by contact activation of locomotion and chemotaxis

Despite their central role in multicellular organization, navigation rules that dictate cell rearrangement remain much to be elucidated. Contact between neighboring cells and diffusive attractant molecules are two of the major determinants of tissue-level patterning, however in most cases, molecular and developmental complexity hinders one from decoding the exact governing rules of individual cell movement. A primordial example of tissue patterning by cell rearrangement is found in the social amoeba Dictyostelium discoideum where the organizing center or the tip self-organize as a result of sorting of differentiating prestalk and prespore cells. Due to its relatively simple and conditional multicellularity, the system provides a rare case where the process can be fully dissected into individual cell behavior. By employing microfluidics and microsphere-based manipulation of navigational cues at the single-cell level, here we uncovered a previously overlooked mode of Dictyostelium cell migration that is strictly directed by cell-cell contact. The cell-cell contact signal is mediated by E-set Ig-like domain containing heterophilic adhesion molecules TgrB1/TgrC1 that act in trans to induce plasma membrane recruitment of SCAR complex and formation of dendritic actin networks, and the resulting cell protrusion competes with those induced by chemoattractant cAMP. Furthermore, we demonstrate that both prestalk and prespore cells can protrude towards the contact signal as well as to chemotax towards cAMP, however when given both signals, prestalk cells orient towards the chemoattractant whereas prespore cells choose the contact signal. These data suggest a new model of cell sorting by competing juxtacrine and diffusive cues each with potential to drive its own mode of collective cell migration. The present findings not only resolve the long standing question of how cells sort in Dictyostelium but also cast light on the remarkable parallels in collective cell migration that evolved independently in metazoa and amoebozoa.

cell biology↗