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. 

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