Cell crawling is central to physiological processes like wound healing, maintenance of tissue, differentiation, development (morphogenesis) and cancer metastasis. How a crawling cell positions its nucleus as it executes its normal tasks of protrusion, adhesion, and retraction of the trailing edge is poorly understood. We propose two specific aims:
Aim 1 : Explain how the nucleus is positioned centrally through generation of mechanical forces on the nuclear surface in crawling fibroblasts.
Aim 2 : Explain how the mechanical linkages between the nucleus and the F-actin cytoskeleton mediate directional persistence of fibroblast crawling. This proposal is innovative for the following reasons: 1) The majority of in vitro studies on nuclear motion have been performed in the context of motility at the edge of a wounded cell monolayer with an emphasis on initial polarization events (where the nucleus is pushed away from the leading edge). In contrast, this study will determine how an isolated crawling cell translates its nucleus in the direction of cell crawling. 2) We work from th novel conceptual view that nuclear positioning is a result of a balance of competing forward and rearward forces which can be pushing or pulling. 3) We propose to use a combination of engineering and biomolecular tools to perturb this nuclear force balance and deduce the direction and relative magnitude of the dominant cytoskeletal forces driving nuclear positioning. The medical significance of this work is due to the fact that abnormal nuclear-cytoskeletal force transfer is thought to be involved in a number of diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, Hutchinson-Gilford progeria syndrome and Dunnigan-type familial partial lipodystrophy. Although the molecular mechanism underlying these diseases remains unclear, it has been hypothesized that these diseases may result (in part) due to abnormal force transmission from the cytoskeleton to the nucleus. Our approach in this proposal relies on mutating nuclear- cytoskeletal linkers that are associated with these diseases and examining alterations in the force balance. This work therefore has both scientific and medical significance.

Public Health Relevance

We seek to understand how cytoskeletal forces are transmitted to the nucleus, and how this force transmission mediates normal cell crawling. This knowledge will help treat the class of diseases termed as nuclear envelopathies (examples include Emery Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome) which are associated with abnormal force generation on the nucleus.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM102486-03
Application #
8703134
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Lyster, Peter
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Florida
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Neelam, Srujana; Dickinson, Richard B; Lele, Tanmay P (2016) New approaches for understanding the nuclear force balance in living, adherent cells. Methods 94:27-32
Mehus, Aaron A; Anderson, Ruthellen H; Roux, Kyle J (2016) BioID Identification of Lamin-Associated Proteins. Methods Enzymol 569:3-22
Kim, Dae In; Jensen, Samuel C; Noble, Kyle A et al. (2016) An improved smaller biotin ligase for BioID proximity labeling. Mol Biol Cell 27:1188-96
Neelam, Srujana; Hayes, Peter Robert; Zhang, Qiao et al. (2016) Vertical uniformity of cells and nuclei in epithelial monolayers. Sci Rep 6:19689
Kent, Ian A; Rane, Parag S; Dickinson, Richard B et al. (2016) Transient Pinning and Pulling: A Mechanism for Bending Microtubules. PLoS One 11:e0151322
Kim, Dae In; Roux, Kyle J (2016) Filling the Void: Proximity-Based Labeling of Proteins in Living Cells. Trends Cell Biol 26:804-817
Kent, Ian A; Lele, Tanmay P (2016) Microtubule-based force generation. Wiley Interdiscip Rev Nanomed Nanobiotechnol :
Aggarwal, Varun; Dickinson, Richard B; Lele, Tanmay P (2016) Concentration Sensing by the Moving Nucleus in Cell Fate Determination: A Computational Analysis. PLoS One 11:e0149213
Torbati, Mehdi; Lele, Tanmay P; Agrawal, Ashutosh (2016) Ultradonut topology of the nuclear envelope. Proc Natl Acad Sci U S A 113:11094-11099
Torbati, Mehdi; Lele, Tanmay P; Agrawal, Ashutosh (2016) An unresolved LINC in the nuclear envelope. Cell Mol Bioeng 9:252-257

Showing the most recent 10 out of 21 publications