Tani Tomomi

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Pectin chemistry and cellulose crystallinity govern pavement cell morphogenesis in a multi-step mechanism

(American Society of Plant Biologists, 2019-08-29) Altartouri, Bara ; Bidhendi, Amir J. ; Tani, Tomomi ; Suzuki, Johnny ; Conrad, Christina ; Chebli, Youssef ; Liu, Na ; Karunakaran, Chithra ; Scarcelli, Giuliano ; Geitmann, Anja

Simple plant cell morphologies, such as cylindrical shoot cells, are determined by the extensibility pattern of the primary cell wall, which is thought to be largely dominated by cellulose microfibrils, but the mechanism leading to more complex shapes, such as the interdigitated patterns in the epidermis of many eudicotyledon leaves, is much less well understood. Details about the manner in which cell wall polymers at the periclinal wall regulate the morphogenetic process in epidermal pavement cells and mechanistic information about the initial steps leading to the characteristic undulations in the cell borders are elusive. Here, we used genetics and recently developed cell mechanical and imaging methods to study the impact of the spatio-temporal dynamics of cellulose and homogalacturonan pectin distribution during lobe formation in the epidermal pavement cells of Arabidopsis (Arabidopsis thaliana) cotyledons. We show that nonuniform distribution of cellulose microfibrils and demethylated pectin coincides with spatial differences in cell wall stiffness but may intervene at different developmental stages. We also show that lobe period can be reduced when demethyl-esterification of pectins increases under conditions of reduced cellulose crystallinity. Our data suggest that lobe initiation involves a modulation of cell wall stiffness through local enrichment in demethylated pectin, whereas subsequent increase in lobe amplitude is mediated by the stress-induced deposition of aligned cellulose microfibrils. Our results reveal a key role of noncellulosic polymers in the biomechanical regulation of cell morphogenesis.
Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy

(American Society for Cell Biology, 2017-08-23) Imai, Ryosuke ; Nozaki, Tadasu ; Tani, Tomomi ; Kaizu, Kazunari ; Hibino, Kayo ; Ide, Satoru ; Tamura, Sachiko ; Takahashi, Koichi ; Shribak, Michael ; Maeshima, Kazuhiro

In eukaryotic cells, highly condensed inactive/silenced chromatin has long been called “heterochromatin.” However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells. Here, using orientation-independent differential interference contrast (OI-DIC) microscopy, which is capable of mapping optical path differences, we investigated the density of the total materials in pericentric foci, a representative heterochromatin model, in live mouse NIH3T3 cells. We demonstrated that the total density of heterochromatin (208 mg/ml) was only 1.53-fold higher than that of the surrounding euchromatic regions (136 mg/ml) while the DNA density of heterochromatin was 5.5- to 7.5-fold higher. We observed similar minor differences in density in typical facultative heterochromatin, the inactive human X chromosomes. This surprisingly small difference may be due to that nonnucleosomal materials (proteins/RNAs) (∼120 mg/ml) are dominant in both chromatin regions. Monte Carlo simulation suggested that nonnucleosomal materials contribute to creating a moderate access barrier to heterochromatin, allowing minimal protein access to functional regions. Our OI-DIC imaging offers new insight into the live cellular environments.
Postnatal structural development of mammalian basilar membrane provides anatomical basis for the maturation of tonotopic maps and frequency tuning

(Nature Research, 2021-04-07) Tani, Tomomi ; Koike-Tani, Maki ; Tran, Mai Thi ; Shribak, Michael ; Levic, Snezana

The basilar membrane (BM) of the mammalian cochlea constitutes a spiraling acellular ribbon that is intimately attached to the organ of Corti. Its graded stiffness, increasing from apex to the base of the cochlea provides the mechanical basis for sound frequency analysis. Despite its central role in auditory signal transduction, virtually nothing is known about the BM’s structural development. Using polarized light microscopy, the present study characterized the architectural transformations of freshly dissected BM at time points during postnatal development and maturation. The results indicate that the BM structural elements increase progressively in size, becoming radially aligned and more tightly packed with maturation and reach the adult structural signature by postnatal day 20 (P20). The findings provide insight into structural details and developmental changes of the mammalian BM, suggesting that BM is a dynamic structure that changes throughout the life of an animal.
Flexible and dynamic nucleosome fiber in living mammalian cells

(Landes Bioscience, 2013-08-12) Nozaki, Tadasu ; Kaizu, Kazunari ; Pack, Chan-Gi ; Tamura, Sachiko ; Tani, Tomomi ; Hihara, Saera ; Nagai, Takeharu ; Takahashi, Koichi ; Maeshima, Kazuhiro

Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.
Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

(American Society for Cell Biology, 2021-01-28) Ishii, Hirokazu ; Tani, Tomomi

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca2+] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.
Polarized light microscopy in reproductive and developmental biology

( 2013-05) Koike-Tani, Maki ; Tani, Tomomi ; Mehta, Shalin B. ; Verma, Amitabh ; Oldenbourg, Rudolf

The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. Therefore, it is a powerful tool to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on chromosome packing in sperm head, first zygote division of the sea urchin, and differentiation initiated by the first uneven cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end by reporting first results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12 through 20.
Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.

(Rockefeller University Press, 2019-03-01) Nagashima, Ryosuke ; Hibino, Kayo ; Ashwin, S. S. ; Babokhov, Michael ; Fujishiro, Shin ; Imai, Ryosuke ; Nozaki, Tadasu ; Tamura, Sachiko ; Tani, Tomomi ; Kimura, Hiroshi ; Shribak, Michael ; Kanemaki, Masato T. ; Sasai, Masaki ; Maeshima, Kazuhiro

Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.
Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

(Rockefeller University Press, 2012-11-26) Li, Wenjing ; Moriwaki, Takashi ; Tani, Tomomi ; Watanabe, Takashi ; Kaibuchi, Kozo ; Goshima, Gohta

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215msps polymerase and the EB1–Sentin duo.
Septin assemblies form by diffusion-driven annealing on membranes

( 2013-12) Bridges, Andrew A. ; Zhang, Huaiying ; Mehta, Shalin B. ; Occhipinti, Patricia ; Tani, Tomomi ; Gladfelter, Amy S.

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knit together into higher-order assemblies. Using fluorescence correlation spectroscopy (FCS), we determined that cytosolic septins are in small complexes suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence (TIRF) microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends and do not exchange subunits in the middle of filaments. This work shows for the first time that annealing is an intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.
Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

(Nature Publishing Group, 2017-12-11) Nordenfelt, Pontus ; Moore, Travis I. ; Mehta, Shalin B. ; Kalappurakkal, Joseph Mathew ; Swaminathan, Vinay ; Koga, Nobuyasu ; Lambert, Talley J. ; Baker, David ; Waters, Jennifer C. ; Oldenbourg, Rudolf ; Tani, Tomomi ; Mayor, Satyajit ; Waterman, Clare M. ; Springer, Timothy

Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.
A composition-dependent molecular clutch between T cell signaling condensates and actin

(eLife Sciences Publications, 2019-07-03) Ditlev, Jonathon ; Vega, Anthony R ; Köster, Darius Vasco ; Su, Xiaolei ; Tani, Tomomi ; Lakoduk, Ashley M ; Vale, Ronald D. ; Mayor, Satyajit ; Jaqaman, Khuloud ; Rosen, Michael K.

During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments. We found that basic regions of Nck and N-WASP/WASP promote association and co-movement of LAT condensates with actin, indicating conversion of weak individual affinities to high collective affinity upon phase separation. Condensates lacking these components were propelled differently, without strong actin adhesion. In cells, LAT condensates lost Nck as radial actin transitioned to the concentric network, and engineered condensates constitutively binding actin moved aberrantly. Our data show that Nck and WASP form a clutch between LAT condensates and actin in vitro and suggest that compositional changes may enable condensate movement by distinct actin networks in different regions of the IS. https://doi.org/10.7554/eLife.42695.001
Radial alignment of microtubules through tubulin polymerization in an evaporating droplet

(Public Library of Science, 2020-04-10) Keya, Jakia Jannat ; Kudoh, Hiroki ; Kabir, Arif Md. Rashedul ; Inoue, Daisuke ; Miyamoto, Nobuyoshi ; Tani, Tomomi ; Kakugo, Akira ; Shikinaka, Kazuhiro

We report the formation of spherulites from droplets of highly concentrated tubulin solution via nucleation and subsequent polymerization to microtubules (MTs) under water evaporation by heating. Radial alignment of MTs in the spherulites was confirmed by the optical properties of the spherulites observed using polarized optical microscopy and fluorescence microscopy. Temperature and concentration of tubulins were found as important parameters to control the spherulite pattern formation of MTs where evaporation plays a significant role. The alignment of MTs was regulated reversibly by temperature induced polymerization and depolymerization of tubulins. The formation of the MTs patterns was also confirmed at the molecular level from the small angle X-ray measurements. This work provides a simple method for obtaining radially aligned arrays of MTs.
Dynamic organization of chromatin domains revealed by super-resolution live-dell imaging

( 2017-06) Nozaki, Tadasu ; Imai, Ryosuke ; Tanbo, Mai ; Nagashima, Ryosuke ; Tamura, Sachiko ; Tani, Tomomi ; Joti, Yasumasa ; Tomita, Masaru ; Hibino, Kayo ; Kanemaki, Masato T. ; Wendt, Kerstin S.

The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy, PALM) and single nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ~160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome–nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
Living cells and dynamic molecules observed with the polarized light microscope : the legacy of Shinya Inoué

(Marine Biological Laboratory, 2016-08) Tani, Tomomi ; Shribak, Michael ; Oldenbourg, Rudolf

In 1948, Shinya Inoué arrived in the United States for graduate studies at Princeton. A year later he came to Woods Hole, starting a long tradition of summer research at the Marine Biological Laboratory (MBL), which quickly became Inoué's scientific home. Primed by his Japanese mentor, Katsuma Dan, Inoué followed Dan's mantra to work with healthy, living cells, on a fundamental problem (mitosis), with a unique tool set that he refined for precise and quantitative observations (polarized light microscopy), and a fresh and brilliant mind that was unafraid of challenging current dogma. Building on this potent combination, Inoué contributed landmark observations and concepts in cell biology, including the notion that there are dynamic, fine structures inside living cells, in which molecular assemblies such as mitotic spindle fibers exist in delicate equilibrium with their molecular building blocks suspended in the cytoplasm. In the late 1970s and 1980s, Inoué and others at the MBL were instrumental in conceiving video microscopy, a groundbreaking technique which married light microscopy and electronic imaging, ushering in a revolution in how we know and what we know about living cells and the molecular mechanisms of life. Here, we recount some of Inoué's accomplishments and describe how his legacy has shaped current activities in polarized light imaging at the MBL.

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