Suzuki Lab
McArdle Laboratory for Cancer Research

Current Research

1. Chromatin, kinetochore, and cell cycle projects

Project 1 : Mechanobiology in chromosome segregation

Chromosome segregation is driven by forces exerted at kinetochores, the specialized protein complexes on chromosomes. These piconewton-scale forces arise from the dynamic interactions between microtubules and kinetochores. Despite their minute magnitude, these forces coordinate the intricate processes of chromosome alignment, organizing mitotic chromosomes of varying sizes into the metaphase plate, and ensuring accurate partitioning of chromosomes into two daughter cells during anaphase. Furthermore, the tension at kinetochores induces both inter- and intra-kinetochore deformation (stretching), which stabilizes kinetochore-microtubule attachments and regulates the spindle assembly checkpoint (SAC). Utilizing super-resolution microscopy and mechanobiological optics, we visualize and quantify these forces with precision, ensuring the fidelity of chromosome segregation.

Schematic of Ndc80 FRET tension biosensor

Project 1 : Visualize and Analyze “Force” at Kinetochores

Related publications:

Project 2 : Kinetochore Functions in Faithful Chromosome Segregation

The kinetochore is a large macromolecular protein complex composed of at least 26 core kinetochore proteins during metaphase, along with additional corona proteins, often referred to as spindle assembly checkpoint (SAC) proteins, which are recruited during prometaphase or at unattached kinetochores. Core kinetochore proteins are classified into at least two categories: inner kinetochore and outer kinetochore proteins.The inner kinetochore proteins, collectively known as the Constitutive Centromere-Associated Network (CCAN), remain associated with centromeric chromatin throughout the cell cycle. Key components of CCAN include CENP-A, CENP-C, the CENP-T complex, and the CENP-H/I complex. In contrast, the outer kinetochore proteins, which constitute the highly conserved KMN network (comprising Knl1, the Mis12 complex, and the Ndc80 complex), are assembled at kinetochores specifically during mitosis. Our research focuses on elucidating the roles of kinetochore proteins in ensuring accurate chromosome segregation. We employ quantitative microscopy, high spatiotemporal resolution imaging, and advanced cell biological approaches to dissect their molecular functions and regulatory mechanisms.

Selected publications:

FRET image

Project 2: Kinetochore Functions in Faithful Chromosome Segregation

Project 3: Kinetochore/Centromere Integrity and Cancer

Failure of accurate chromosome segregation causes CIN (Chromosomal INstability), which includes occurrences of aneuploidy, chromosome breakage, and micronuclei. CIN leads to cancer, various birth defects, as well as up to one third of miscarriage, lethality, and infertility cases. Aneuploidy is particularly important because it is a hallmark of cancer, especially poor prognosis cancer, as ~90 % of solid tumors show aneuploidy. A major cause of aneuploidy is kinetochore-microtubule attachment errors. One of these errors is lagging chromosome, a kind of “Tug of War” within a sister kinetochore pair by improper microtubule assembly and is often found during anaphase. Important kinetochore functions include monitoring mitotic errors and error corrections; thus, it is critical to study how the kinetochore ensures the integrity of chromosome segregation. We are studying the mechanism of how the loss of kinetochore integrity causes cancer with a focus on the kinetochore functions, the architectural integrity of kinetochores, and the regulation of kinetochore proteins/genes.

Related publications:

FRET image of lagging chromosome

2. Virology projects

Project 1: HIV-1 cell cycle projects

HIV-1 Vif (Viral Infectivity Factor) and Vpr (Viral Protein R) are well-established inducers of cell cycle arrest in host cells. The cell division cycle, critical for cell proliferation, is composed of distinct phases: G0, G1, S, G2, and M. While previous studies have reported that Vif and Vpr arrest cells in the G2/M phases using traditional flow cytometry, this method measures relative DNA content and cannot reliably distinguish between G2 and M phases. In our recent study, we demonstrated that Vif induces a unique and robust pseudo-metaphase arrest, as revealed by high-spatiotemporal resolution long-term live-cell imaging. This advanced approach provides single-cell resolution and unparalleled accuracy in identifying specific cell cycle stages. We are currently investigating the molecular mechanisms underlying the cell cycle arrests induced by Vif and Vpr, as well as their functional roles in the HIV-1 viral life cycle.

Related publications:

 

Project 2: HIV-1 cell RNA tracking projects

HIV-1 genomic RNA (gRNA) forms dimers within HIV-1 virions, a process essential for viral infectivity. Following HIV-1 infection, the viral gRNA integrates into the host genome. During the lytic phase, replicated gRNAs are transported to the cytoplasm and subsequently localize to the plasma membrane, where HIV-1 capsid assembly occurs to produce infectious virions. However, the precise mechanisms and intracellular sites of HIV-1 gRNA dimerization within host cells remain elusive. By employing self-fluorescent tagging of HIV-1 gRNA in combination with advanced super-resolution microscopy techniques, we aim to visualize individual gRNA molecules and unravel the spatiotemporal dynamics underlying HIV-1 gRNA dimerization.

Project 3: Spatiotemporal regulation of Epstein-Barr virus (EBV) capsid morphogenesis

Epstein-Barr virus (EBV) infects ~90% of adults and causes infectious mononucleosis as well as several lymphomas and carcinomas. EBV has two stages of infection, latent and lytic phases. EBV produces infectious virions in lytic phase, whereas EBV is maintained as closed plasmids without producing virions during latent phase. Therefore, it is important to understand the regulations of lytic phase for preventing EBV infection. The size of EBV is ~100 nm, that makes difficult to study using fluorescence microscopy. We developed a novel super-resolution microscopy method, 12x3D-Expansion Microscopy (12x 3D-ExM), which achieves ~15 nm resolution  and  successfully visualizes a single EBV.  We are investigating e the mechanism underlying capsid assembly, that contributes to identify therapeutic targets to prevent/treat infection and improve patient outcomes.

Related publications:

3. Super-resolution microscopy, Optics, and image software development projects

Project 1: Image Analysis Software development

Fluorescence microscopy serves as a cornerstone of modern cell biology research, offering unparalleled insights into cellular structures and processes. Our focus lies in pioneering advanced image analysis software including deep -learning tailored to super-resolution microscopy, quantitative image analysis, and the exploration of cell cycle dynamics.

Related publications:

 

Project 2: Super-resolution microscopy development

Fluorescence microscopy is an indispensable tool in cell biology, yet its resolution is fundamentally constrained by the diffraction limit to approximately 250 nm laterally and 600 nm axially. Since numerous cellular structures are smaller than these limits, super-resolution microscopy techniques, which break the optical resolution barrier, have been developed. In our research, we focus on advancing novel super-resolution microscopy methods and optimizing labeling protocols for diverse biological specimens. For instance, we recently developed optimized expansion microscopy (ExM) techniques, termed 4x and 12x 3D-ExM. ExM is an innovative super-resolution technology that achieves super-resolution by physically expanding the hydrogel-embedded specimen. Theoretically, ExM improves resolution proportionally to the expansion factor. Our 12x 3D-ExM method enables isotropic expansion of specimens by 12-fold in a single-step process, achieving  ~15 nm lateral and ~40 nm axial resolution using standard confocal microscopy, without the need for post-image processing. This approach offers novel insights into cellular architecture at nanoscale resolution.

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