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Shrinkage, Swelling and Macromolecular Crowding in Cell Death

Rana, Priyanka Shailendra
http://orcid.org/0000-0001-5774-4383
http://rave.ohiolink.edu/etdc/view?acc_num=kent1595850511516452

Abstract Details

2020, PHD, Kent State University, College of Arts and Sciences / Department of Biological Sciences.
Cell shrinkage and dehydration are significant, essential, and most reproducible characteristics of apoptosis, Dehydration and fragmentation are the two general mechanisms which are responsible for apoptotic volume decrease (AVD). However, previous authors have not been able to differentiate between these two very different processes and tended to attribute the entire volume decrease to water loss. Based on some theoretical considerations, we believe that such interpretation cannot be correct in all cases. We are now equipped to quantify dehydration and fragmentation and investigate their regulation separately. But to do that, we had to develop new experimental methods. We first developed a method based on calibrated transport of intensity equation (TIE) microscopy combined with transmission-through dye (TTD) microscopy previously developed in our lab. The combination of TTD and TIE allows us to differentiate dehydration from fragmentation and we have demonstrated in various systems that our method works. Second, because intracellular water balance depends on monovalent ions, we had to improve the existing methods for quantification of intracellular ions. That involved some highly original approaches. We developed a method for intracellular potassium measurement. This approach used to calibrate the potassium sensitive fluorescent probe can be further used to quantify intracellular sodium and chloride. These methods enable us to undertake rigorous and quantitative studies of volume and water behavior in various systems where cells undergo apoptotic or necrotic death. We have been able to uncover and explain new paradoxical features of necrotic membrane blebs. We showed that water measurement reveals new features of long-term volume maintenance in anisosmotic environment. We showed that water loss induced by the same apoptotic agent (staurosporine) depends on cell type and attachment. We have been able to circumvent an inherent limitation of many previous studies that could not distinguish between individual roles of potassium loss and shrinkage in apoptosis development. Our current data demonstrate that water loss is an active proapoptotic factor in its own right, while potassium concentration has a minor importance. We present evidence that persistent shrinkage can cause apoptosis regardless of intracellular sodium/potassium composition or the state of actin cytoskeleton, suggesting that an increase in protein density (macromolecular crowding) acts as a direct apoptotic factor; (2) strong potassium dependence of caspase activation is only observed in swollen cells with reduced density of cytosolic proteins.
Michael Model (Advisor)
Derek Damron (Committee Member)
Gail Fraizer (Committee Member)
Soumitra Basu (Committee Member)
Bansidhar Datta (Committee Member)
195 p.
Biochemistry; Biology; Biophysics; Cellular Biology; Molecular Biology; Physiology
Macromolecular crowding; Cell volume; Volume sensing; Apoptotic shrinkage; Necrotic swelling; Ion probes; Ionophores; Intracellular potassium measurement; Quantitative phase imaging; Transmission-through dye imaging, Transport-of-intensity equation

Recommended Citations

Citations

  • Rana, P. S. (2020). Shrinkage, Swelling and Macromolecular Crowding in Cell Death [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1595850511516452

    APA Style (7th edition)

  • Rana, Priyanka Shailendra. Shrinkage, Swelling and Macromolecular Crowding in Cell Death. 2020. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1595850511516452.

    MLA Style (8th edition)

  • Rana, Priyanka Shailendra. "Shrinkage, Swelling and Macromolecular Crowding in Cell Death." Doctoral dissertation, Kent State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=kent1595850511516452

    Chicago Manual of Style (17th edition)

kent1595850511516452
411
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This open access ETD is published by Kent State University and OhioLINK.
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