A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division

Virtual Reality and Well-Being in Older Adults: Results from a Pilot Implementation of Virtual Reality in Long-Term Care

This paper describes the findings of a pilot implementation project that explored the potential of virtual reality (VR) technology in recreational programming to support the well-being of older adults in long-term care (LTC) homes

Implementation of a Virtual Reality Recreation Program in Long-term Care

Introduction: This manuscript describes the implementation of a Virtual Reality (VR) recreation program at long-term care sites across Ontario, Canada, using the RE-AIM Framework to guide the implementation and its evaluation. Methods: We developed a VR recreation program to enhance the lives of long-term care residents, through 3 sequential phases. In Phase 1, we learned about resident and staff needs through focus groups, staff surveys and observations. In Phase 2, we developed 10 VR experiences, based on the data from Phase 1. In Phase 3, we implemented the VR experiences and supporting manual and measured their implementation, using the RE-AIM Framework. Results: We found the VR program to be highly (but not consistently) implementable across all sites. Factors that supported implementation were the following: resident interest in the content and technology, relative ease of use for staff to implement and formally integrating VR into the recreation calendar. Factors that impeded implementation were the following: the size of the headset, inability for the headset to cast given the sites’ Information Technology infrastructure and some content that was not engaging. Conclusions: VR programs are highly implementable and this implementation is enhanced by integration of the program into existing recreational systems, ease of use and resident engagement

Decoding protein kinase signaling during mitosis: Exploiting molecular scaffolds and developing drug-targeting tools for studying local kinase biology

Thesis (Ph.D.)--University of Washington, 2020Fundamental cellular processes such as cell division, migration, differentiation, and growth require environmental signals to be converted into chemical responses that generate biological outputs. Accordingly, these events rely on the precise and timely regulation of signaling proteins inside the cell. Organization of enzymes in space and time becomes particularly important in a process such as mitosis where regulation of mitotic protein kinases ensures that DNA is properly segregated between the two daughter cells. During mitosis, two key protein kinases, Aurora A and polo-like kinase 1 (Plk1) carry out many functions to ensure the fidelity of cell division. Current approaches in which small-molecule kinase inhibitor drugs are used to ascribe function to Aurora A and Plk1 provide important insight into the global roles of these proteins inside the cell. However, since these enzymes share similar localization patterns and many overlapping roles, knowing their individual contributions at specific locations is not possible with existing pharmacological approaches. This is because traditional inhibitor drugs distribute throughout the cell, inhibiting enzymes at many locations, and thus limit the ability to resolve how a protein functions at a distinct organelle. Therefore, decoding the roles of Aurora A and Plk1 at distinct subcellular locations is critical for understanding how cell division is regulated. More importantly, elucidating local kinase action can uncover how signaling becomes dysregulated in disease, thus paving the way for development of more effective therapeutics. Using a combination of chemical genetics, gene-editing, super-resolution microscopy, live-cell imaging, and biochemistry the studies in this thesis uncover how Aurora A and Plk1 coordinate signaling events at distinct locations in the cell during mitosis. In this work, I exploit the local scaffolding ability of Gravin, an A-kinase anchoring protein (AKAP), to study how anchored pools of Aurora A and Plk1 facilitate mitotic events. Additionally, I develop a new drug-targeting system called LoKI (Local Kinase Inhibition) to deliver Aurora A and Plk1 inhibitors to specific subcellular locations to further probe local kinase action. My studies uncover that Gravin localizes active pools of Aurora A and Plk1 at mitotic centrosomes and show that loss of local kinase activity at this location attenuates substrate phosphorylation, produces mitotic spindle defects, and prolongs mitosis. I also discover a new role for this anchoring protein in coordinating the recruitment of γ-tubulin during mitosis and demonstrate that loss of Gravin disrupts protein-protein interactions that facilitate the proper targeting of this key microtubule nucleating component. Together, these studies elucidate how local kinase action drives mitosis and provide evidence that subcellular targeting of protein kinases is a molecular mechanism that underlies precise execution of critical protein phosphorylation events inside cells

A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells

Abstract Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin −/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division

A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division