Orientation-independent-DIC imaging reveals that a transient rise in depletion attraction contributes to mitotic chromosome condensation.

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion attraction/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using an orientation-independent-differential interference contrast module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prophase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like. These results suggest that a transient rise in depletion attraction, likely triggered by the relocation of macromolecules (proteins, RNAs, and others) via nuclear envelope breakdown and by a subsequent decrease in cell volumes, contributes to mitotic chromosome condensation, shedding light on a different aspect of the condensation mechanism in living human cells.

Leadless Pacemaker Implantation in Severe Kyphosis: Challenging Implantation for Tortuous IVC and Narrow RA.

Leadless pacemaker implantation is recognized as safe and effective for treating bradycardia. However, there are limited descriptions of its use in patients with complex anatomical considerations. Here, we present a case detailing the successful implantation of a leadless pacemaker with a tortuous inferior vena cava and a narrow right atrium.

Live-cell imaging under centrifugation characterized the cellular force for nuclear centration in the Caenorhabditis elegans embryo.

Organelles in cells are appropriately positioned, despite crowding in the cytoplasm. However, our understanding of the force required to move large organelles, such as the nucleus, inside the cytoplasm is limited, in part owing to a lack of accurate methods for measurement. We devised a novel method to apply forces to the nucleus of living, wild-type Caenorhabditis elegans embryos to measure the force generated inside the cell. We utilized a centrifuge polarizing microscope (CPM) to apply centrifugal force and orientation-independent differential interference contrast (OI-DIC) microscopy to characterize the mass density of the nucleus and cytoplasm. The cellular forces moving the nucleus toward the cell center increased linearly at ~14 pN/µm depending on the distance from the center. The frictional coefficient was ~1,100 pN s/µm. The measured values were smaller than previously reported estimates for sea urchin embryos. The forces were consistent with the centrosome-organelle mutual pulling model for nuclear centration. Frictional coefficient was reduced when microtubules were shorter or detached from nuclei in mutant embryos, demonstrating the contribution of astral microtubules. Finally, the frictional coefficient was higher than a theoretical estimate, indicating the contribution of uncharacterized properties of the cytoplasm.

Orientation-Independent-DIC imaging reveals that a transient rise in depletion force contributes to mitotic chromosome condensation.

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion force/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using orientation-independent-differential interference contrast (OI-DIC) module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prometaphase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like, with further condensation. These results suggest that a transient rise in depletion force, likely triggered by the relocation of macromolecules (proteins, RNAs and others) via nuclear envelope breakdown and also by a subsequent decrease in cell-volumes, contributes to mitotic chromosome condensation, shedding light on a new aspect of the condensation mechanism in living human cells.

Detection of Glutamate Encapsulated in Liposomes by Optical Trapping Raman Spectroscopy.

The transmission of neuronal information is propagated through synapses by neurotransmitters released from presynapses to postsynapses. Neurotransmitters released from the presynaptic vesicles activate receptors on the postsynaptic membrane. Glutamate acts as a major excitatory neurotransmitter for synaptic vesicles in the central nervous system. Determining the concentration of glutamate in single synaptic vesicles is essential for understanding the mechanisms of neuronal activation by glutamate in normal brain functions as well as in neurological diseases. However, it is difficult to detect and quantitatively measure the concentration of glutamate in single synaptic vesicles owing to their small size, i.e., ∼40 nm. In this study, to quantitatively evaluate the concentrations of the contents in small membrane-bound vesicles, we developed an optical trapping Raman spectroscopic system that analyzes the Raman spectra of small objects captured using optical trapping. Using artificial liposomes encapsulating glutamate that mimic synaptic vesicles, we investigated whether spontaneous Raman scattered light of glutamate can be detected from vesicles trapped at the focus using optical forces. A 575 nm laser beam was used to simultaneously perform the optical trapping of liposomes and the detection of the spontaneous Raman scattered light. The intensity of Raman scattered light that corresponds to lipid bilayers increased with time. This observation suggested that the number of liposomes increased at the focal point. The number of glutamate molecules in the trapped liposomes was estimated from the calibration curve of the Raman spectra of glutamate solutions with known concentration. This method can be used to measure the number of glutamate molecules encapsulated in synaptic vesicles in situ.

Autistic-Like Behavior and Impairment of Serotonin Transporter and AMPA Receptor Trafficking in N-Ethylmaleimide Sensitive Factor Gene-Deficient Mice.

Autism spectrum disorder (ASD), characterized by profound impairment in social interactions and communication skills, is the most common neurodevelopmental disorder. Many studies on the mechanisms underlying the development of ASD have focused on the serotonergic system; however, these studies have failed to completely elucidate the mechanisms. We previously identified N-ethylmaleimide-sensitive factor (NSF) as a new serotonin transporter (SERT)-binding protein and described its importance in SERT membrane trafficking and uptake in vitro. In the present study, we generated Nsf +/- mice and investigated their behavioral, neurotransmitter, and neurophysiological phenotypes in vivo. Nsf +/- mice exhibited abnormalities in sociability, communication, repetitiveness, and anxiety. Additionally, Nsf loss led to a decrease in membrane SERT expression in the raphe and accumulation of glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors at the synaptic membrane surface in the hippocampal CA1 region. We found that postsynaptic density and long-term depression were impaired in the hippocampal CA1 region of Nsf +/- mice. Taken together, these findings demonstrate that NSF plays a role in synaptic plasticity and glutamatergic and serotonergic systems, suggesting a possible mechanism by which the gene is linked to the pathophysiology of autistic behaviors.

Development of nanobody-based POLArIS orientation probes enabled multi-color/multi-target orientation imaging in living cells.

Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms.

Postnatal structural development of mammalian Basilar Membrane provides anatomical basis for the maturation of tonotopic maps and frequency tuning.

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.

POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs.

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.

Deuterated Glutamate-Mediated Neuronal Activity on Micro-Electrode Arrays.

The excitatory synaptic transmission is mediated by glutamate in neuronal networks of the mammalian brain. In addition to the synaptic glutamate, extra-synaptic glutamate is known to modulate the neuronal activity. In neuronal networks, glutamate uptake is an important role of neurons and glial cells for lowering the concentration of extracellular glutamate and to avoid the excitotoxicity by glutamate. Monitoring the spatial distribution of intracellular glutamate is important to study the uptake of glutamate, but the approach has been hampered by the absence of appropriate glutamate analogs that report the localization of glutamate. Deuterium-labeled glutamate (GLU-D) is a promising tracer for monitoring the intracellular concentration of glutamate, but physiological properties of GLU-D have not been studied. Here we study the effects of extracellular GLU-D for the neuronal activity by using primary cultured rat hippocampal neurons that form neuronal networks on microelectrodes array. The frequency of firing in the spontaneous activity of neurons increased with the increasing concentration of extracellular GLU-D. The frequency of synchronized burst activity in neurons increased similarly as we observed in the spontaneous activity. These changes of the neuronal activity with extracellular GLU-D were suppressed by antagonists of glutamate receptors. These results suggest that GLU-D can be used as an analog of glutamate with equivalent effects for facilitating the neuronal activity. We anticipate GLU-D developing as a promising analog of glutamate for studying the dynamics of glutamate during neuronal activity.

Birefringence Changes of Dendrites in Mouse Hippocampal Slices Revealed with Polarizing Microscopy.

Intrinsic optical signal (IOS) imaging has been widely used to map the patterns of brain activity in vivo in a label-free manner. Traditional IOS refers to changes in light transmission, absorption, reflectance, and scattering of the brain tissue. Here, we use polarized light for IOS imaging to monitor structural changes of cellular and subcellular architectures due to their neuronal activity in isolated brain slices. To reveal fast spatiotemporal changes of subcellular structures associated with neuronal activity, we developed the instantaneous polarized light microscope (PolScope), which allows us to observe birefringence changes in neuronal cells and tissues while stimulating neuronal activity. The instantaneous PolScope records changes in transmission, birefringence, and slow axis orientation in tissue at a high spatial and temporal resolution using a single camera exposure. These capabilities enabled us to correlate polarization-sensitive IOS with traditional IOS on the same preparations. We detected reproducible spatiotemporal changes in both IOSs at the stratum radiatum in mouse hippocampal slices evoked by electrical stimulation at Schaffer collaterals. Upon stimulation, changes in traditional IOS signals were broadly uniform across the area, whereas birefringence imaging revealed local variations not seen in traditional IOS. Locations with high resting birefringence produced larger stimulation-evoked birefringence changes than those produced at low resting birefringence. Local application of glutamate to the synaptic region in CA1 induced an increase in both transmittance and birefringence signals. Blocking synaptic transmission with inhibitors CNQX (for AMPA-type glutamate receptor) and D-APV (for NMDA-type glutamate receptor) reduced the peak amplitude of the optical signals. Changes in both IOSs were enhanced by an inhibitor of the membranous glutamate transporter, DL-TBOA. Our results indicate that the detection of activity-induced structural changes of the subcellular architecture in dendrites is possible in a label-free manner.

Radial alignment of microtubules through tubulin polymerization in an evaporating droplet.

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.

Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism.

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.

A composition-dependent molecular clutch between T cell signaling condensates and actin.

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.

Genetically encoded orientation probes for F-actin for fluorescence polarization microscopy.

Fluorescence polarization microscopy, which can visualize both position and orientation of fluorescent molecules, is useful for analyzing architectural dynamics of proteins in vivo, especially that of cytoskeletal proteins such as actin. Fluorescent phalloidin conjugates and SiR-actin can be used as F-actin orientation probes for fluorescence polarization microscopy, but a lack of appropriate methods for their introduction to living specimens especially to tissues, embryos, and whole animals hampers their applications to image the orientation of F-actin. To solve this problem, we have developed genetically encoded F-actin orientation probes for fluorescence polarization microscopy. We rigidly connected circular permutated green fluorescent protein (GFP) to the N-terminal α-helix of actin-binding protein Lifeact or utrophin calponin homology domain (UtrCH), and normal mEGFP to the C-terminal α-helix of UtrCH. After evaluation of ensemble and single particle fluorescence polarization with the instantaneous FluoPolScope, one of the constructs turned out to be suitable for practical usage in live cell imaging. Our new, genetically encoded F-actin orientation probe, which has a similar property of an F-actin probe to conventional GFP-UtrCH, is expected to report the 3D architecture of the actin cytoskeleton with fluorescence polarization microscopy, paving the way for both the single molecular orientation imaging in cultured cells and the sub-optical resolution architectural analysis of F-actin networks analysis of F-actin in various living systems.

Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.

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.

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration.

Integrin αß 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 ß-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.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions.

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVß3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.

Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy.

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.