The amyloid precursor protein (APP) has been intensely studied for its role in Alzheimer's disease, but its physiological function remains unclear. In neurons, APP and its homologs, the amyloid precursor-like proteins (APLPs) are present at synapses and promote synaptogenesis. Astrocytes also express APP although a role for astrocytic APP has not been fully explored. We have studied the expression and function of APP in rodent astrocytes in vitro and in vivo. shRNA-mediated knockdown of astrocytic APP compromises astrocyte morphological elaboration in hippocampal cultures and in the intact brain. Our results highlight a role of astrocytic APP and possibly of APLPs in shaping astrocyte morphological complexity. We are currently examining how astrocytic APP affects the dynamics of tripartite synapses.

Three-dimensional (3D) structural analysis is crucial to investigate the structural and functional properties of nanoparticles. Transmission electron microscopy (TEM) is a widely used technique to perform such characterization, however, conventional TEM images only provide two-dimensional projections of the 3D object examined. Here we propose a novel core-towards-surface 3D reconstruction strategy based on methods used in single-particle cryo-EM to reconstruct an average 3D model of 18±2 nm gold nanoparticles by starting from a 3 nm core, and expanding the reconstruction stepwise towards the surface of the nanoparticle. Our tailored approach enabled us to reconstruct the entire volume of the nanoparticle at the final resolution of 0.86 Å. The excellent agreement between the experimental 3D reconstruction and a theoretical map calculated by quenched molecular dynamics demonstrates that our method is suitable to provide statistically relevant 3D structures of nanoparticles which can be subsequently used to perform ensemble analysis for strain mapping or to determine the lattice parameter of alloyed nanoparticles of different compositions. 

Microfluidics and lab-on-a-chip devices have become powerful platforms for manipulating fluids at small scales, significantly advancing biophysics and biotechnology research. In this talk, I will present two examples of using microfluidics in microbial population genetics and disease diagnosis. The first example involves a microfluidic device with a controlled microenvironment designed to study population genetics, where microbial populations proliferate in small channels. In these environments, reproducing cells organize into parallel lanes, and as they shift, they can potentially expel other cells from the channel. By combining theoretical models and experiments, we found that genetic diversity is rapidly lost along these lanes. Specifically, our experiments demonstrated that a population of proliferating Escherichia coli in a microchannel organizes into lanes of genetically identical cells within just a few generations. The second example highlights the development of advanced microfluidic sensing platforms for rapid and sensitive detection of biomarkers. One such platform employs an optomicrofluidic approach, utilizing localized surface plasmon resonance (LSPR) with gold nanospikes fabricated by electrodeposition within a microfluidic device, coupled with an optical probe, to detect antibodies against the SARS-CoV-2 spike protein in diluted human plasma with a detection limit of approximately 0.5 pM (0.08 ng/mL) within 30 minutes. Additionally, recent work by Mazzaracchio et al. (2023) demonstrates the potential of a duplex electrochemical microfluidic sensor in distinguishing between natural and vaccine-induced humoral responses. Moreover, the versatility of microfluidic platforms extends beyond infectious disease diagnostics, as shown by Funari et al. (2024), who developed a multiplexed opto-microfluidic biosensing platform for the detection of prostate cancer biomarkers. These examples collectively underscore the broad applicability and efficacy of microfluidic technologies in advancing diagnostics across various fields. 

Cytoplasmic dynein is a well-conserved microtubule-based motor that transports cargo molecules toward microtubule minus-end to spatially organize intracellular structure. Recent studies established that dynein itself is auto-inhibited but activated as a highly processive motor by interacting with dynactin and activating adaptors. During mitosis, dynein interacts with dynactin and NuMA, and focuses microtubule minus-ends at spindle poles to promote bipolar spindle assembly. The dynein-dynactin-NuMA (DDN) complex is also assembled at the cell cortex to capture and pull on astral microtubule plus-ends for spindle positioning. However, how these DDN complexes are spatiotemporally regulated to perform distinct functions remains poorly understood. In this talk, we will show our recent studies and discuss how the DDN complexes are regulated at spindle poles and the cell cortex in somatic human cells. In addition, we will discuss whether and how dynein complexes perform specialized functions for spindle assembly and positioning in extremely large vertebrate embryos. 

I am going briefly cover two topics in my talk.  

First, I would like to talk about development of an ultrafast camera system that enables the highest time resolutions in single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore we identified. Using theoretical frameworks developed for the analysis of single-molecule trajectories in the plasma membrane (PM), this camera successfully detected fast hop diffusion of membrane molecules in the PM, previously detectable only in the apical PM by using less preferable 40-nm gold probes, thus helping to elucidate the principles governing the PM organization and molecular dynamics. Furthermore, as described in the companion paper, this camera allows simultaneous data acquisitions for PALM/dSTORM at as fast as 1 kHz, with 29/19 nm localization precisions in the 640x640 pixel view-field. 

Using our newly-developed ultrafast camera, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. 

Second, I will talk about the liquid nano-platform for signal integration on the PM, called iTRVZ. Crosstalk of cellular signaling pathways is essential for integrating them for inducing coordinated final cell responses. However, how signaling molecules are assembled to induce signal integration remains largely unknown. Here, using advanced single-molecule imaging, we found a nanometer-scale liquid-like platform for integrating the signals downstream from GPI-anchored receptors and receptor-type tyrosine kinases. The platform employs some of the focal adhesion proteins, including integrin, talin, RIAM, VASP, and zyxin, but is distinct from focal adhesions, and is thus termed iTRVZ. The iTRVZ formation is driven by the protein liquid-liquid phase separation and the interactions with the raft domains in the plasma membrane and cortical actin. iTRVZ non-linearly integrates the two distinctly different receptor signals, and thus works as an AND gate and noise filter. Using an in-vivo mouse model, we found that iTRVZ greatly enhances tumor growth. 

Atomic force microscopy (AFM) has been a powerful tool that allows to directly visualize nanoscale dynamics of proteins and DNAs in liquid without labeling. However, such high-resolution AFM imaging typically requires fixation of biological samples onto a solid substrate. Therefore, it has been often questioned if the observed structures or phenomena truly represent those inside living cells. To overcome this problem, we recently developed nanoendoscopy AFM (NE-AFM), where a needle probe is inserted into a living cell to perform intracellular AFM observations1. So far, we have demonstrated 3D imaging of the whole cell structure and actin fibers, and 2D imaging of dynamic structural changes in the actin cortical fibers on the inner surface of the bottom cell membrane. In addition, we demonstrated that these measurements do not cause serious damage to a cell despite the repeated insertion of the probe into the cell.  

  NE-AFM has two distinctive advantages over other imaging techniques. One of them is the capability of nanoscale imaging at intra-cellular interfaces. To take advantage of this, we are investigating dynamics of focal adhesions (FAs). FAs are the intracellular structure connecting between actin fibers and extracellular matrix and play critical roles in cell adhesion and motility. By combining NE-AFM and confocal fluorescent microscope, we simultaneously observed time-lapse changes of the 3D FA structures and paxillin distributions during their growth. From the confocal image, we can identify the position of FAs and perform their 3D-AFM imaging. The 3D-AFM images reveal that the FAs become thicker during their growth. Besides, the actin fiber associated with the FA was initially in contact with the upper cell membrane but detached as the FA grows. This indicates that actin molecules are provided from the cortical actin network during the fiber growth. These detailed nanoscale structural changes are directly captured by NE-AFM in living cells. 

  Another strength of NE-AFM is the capability to measure the nanomechanical properties in living cells. With this capability, we are investigating nuclear envelope (NE) elasticity. NE is supported by lipids, lamins and lamin associated domains (LADs) of chromatins. Among them, LADs are much thicker than others and hence considered to determine the NE elasticity. Meanwhile, LAD organization is considered to be related to the gene expression and related diseases known as nuclear envelopathies. Therefore, there have been great interests in NE elasticity measurements. In addition, nuclear elasticity has attracted attention in cancer research area due to its correlation with cell resistance to the external pressure during its migration and invasion. To address these issues, we use NE-AFM to measure NE elasticity by directly indenting the NE surface with a needle probe. So far, we found that the NE elasticity increases when the serum was depleted and decreases when the EMT was induced by applying TGFb. This is reasonable as the gene expression activity should decrease when the cells are arrested at the G0 phase and increase when the cells become more invasive due to the EMT. These results confirm strong correlation between the NE elasticity and gene expression activity. With this basic understanding, we are now exploring various possibilities of NE-AFM studies on NE elasticity. 

 In my talk, I will introduce recent advancements in scanning ion conductance microscopy (SICM) for live cell imaging. SICM is a type of scanning probe microscopy that uses a glass nanopipette as a probe to achieve nanoscale imaging of sample surfaces in a liquid environment. This technique is particularly advantageous due to its low-invasive measurement principle, allowing for long-term visualization of cellular surfaces. Recent developments in SICM, carried out by several groups including my group [1], have enabled the capture of nanostructural dynamics on the cellular surface at sub-second time scales. Moreover, simultaneous imaging of topography and elasticity has been developed, providing comprehensive understanding of cellular states by combining multiple types of information. 

 Despite these advancements, there is still a need to improve the imaging rate of SICM for broader applications in nanobiosciences. Enhancing accessibility to complex structures, such as fragile tissues, remains an area of ongoing development. In my presentation, I will review recent SICM advancements and discuss the challenges we face in improving spatiotemporal resolution and accessibility to samples with complex architectures. Additionally, I will present our recent work on genotype-defined cancer cells [2], including three-dimensional organoids [3], where SICM has been used to identify quantitative differences in cellular states through physical parameters such as roughness, membrane fluctuation, and local elasticity. 

Human RNA undergoes various modifications, among them, the most prevalent modification is A-to-I RNA editing catalyzed by ADAR (adenosine deaminase acting on RNA). This process involves the conversion of adenosine (A) to inosine (I) in double-stranded RNA regions, which can influence RNA stability, splicing, and protein functions. The aberrant expression of ADAR is associated with various cancers, highlighting its potential as both a prognostic biomarker and a therapeutic target. Our study also revealed that ADAR1 expression was higher in breast cancer tissues compared to normal tissues, suggesting that ADAR1 contributes to cancer proliferation. We found that ADAR1 increased the expression of DHFR (dihydrofolate reductase), a target protein of methotrexate, an anti-cancer drug, by disrupting the binding of miR-25-3p and miR-125-3p, leading to drug resistance. We demonstrated that suppressing ADAR1 expression decreased DHFR level and enhanced the effectiveness of methotrexate, indicating that inhibiting ADAR1 can help overcome resistance to anti-cancer drugs.  

 We screened DNA aptamers against ADAR1 and identified Apt38, an aptamer with high binding affinity for ADAR1. Although its inhibitory effects on A-to-I RNA editing were weak, we leveraged its binding capability to develop an electrochemical biosensor for the precise detection of ADAR1 in biological samples. The biosensor combines Apt38 with an electrochemical transduction method, utilizing a sandwich assay format with specific antibodies and gold nanoparticles. It detects ADAR1 at concentrations as low as 0.53 nM via differential pulse voltammetry (DPV), offering a highly sensitive, cost-effective, and rapid detection method with significant implications for cancer prognosis and monitoring.  

 Limited structural information on ADAR1 complexes has hindered the identification of effective inhibitors. To address this challenge, we employed 3D computational modeling and high-speed atomic force microscopy (HS-AFM) to study the dynamics of ADAR1. We identified key interface regions (IFx and IFy) within the deaminase domain that are crucial for ADAR1 dimerization and observed stable dimeric structures in the presence of substrate dsRNA. Our findings also underscore the role of the flexible N-terminal region in maintaining ADAR1 dimer stability and dynamics. These insights are essential for developing targeted inhibitors to modulate ADAR1 activity, paving the way for new therapeutic interventions.  

 I am going briefly introduce nano imaging of nuclear pore territories in my talk. 

 First, I would like to talk about the different jobs that nuclear pore complexes (NPCs) do. Inside the NPC, an assembly of natively unfolded ("spider cobweb-like") proteins dictates the chemical and size selectivity of transport into and out of the nucleus. NPCs are not only as intracellular supply chain terminals controlling the transport of proteins, NPCs also play critical roles in spindle polarity, the formation of aneuploidies, the growth of colon and brain cancer, and the location of super enhancers at the epigenomic and spatial levels.  

 Second, I would like to talk about NPCs dynamic structures and how viral proteins move towards to NPCs. NPCs restrict free diffusion to molecules below 5 nm while facilitating the active transport of selected cargoes, sometimes as large as the pore itself. This versatility implies an important pore plasticity. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record different viral proteins, microtubules, EVs and how they transport towards nuclear pore from purified proteins, cells, organoids, and mouse brain tissues. 

 Eukaryotic cells are compartmentalized into the nucleus and cytoplasm, with gene expression processes occurring in these distinct compartments—transcription in the nucleus and translation in the cytoplasm. Nonsense-mediated mRNA decay (NMD) is a translation-coupled mRNA decay pathway triggered by premature termination codons (PTCs). Although the recognition of in-frame PTCs occurs exclusively in cytoplasmic ribosomes, unexpected transcriptional alterations in genes with PTCs have been observed. This suggests crosstalk between the nucleus and cytoplasm during gene expression regulation. Addressing cellular events in these separate compartments simultaneously remains challenging. In this presentation, I will discuss a study that employs a real-time imaging technique to simultaneously monitor the transcriptional activity of both wild-type and NMD-targeted reporter genes in individual cells. Our findings reveal that NMD, which operates in the cytoplasm, induces significant transcriptional changes in a PTC-specific manner, providing compelling evidence for a robust connection between cytoplasmic decay and nuclear transcription. Additionally, I will present collaborative research conducted at NanoLSI, highlighting the diverse technologies used in our investigations.