Title

To Be or Not To Be a Protrusion: Unraveling the Determinants of Protrusion Formation

Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Carol Heckman, PhD

Second Advisor

Jeffrey Snyder, PhD (Committee Member)

Third Advisor

Peter Gorsevski, PhD (Committee Member)

Fourth Advisor

Michael Geusz, PhD (Committee Member)

Fifth Advisor

Roudabeh Jamasbi, PhD (Committee Member)

Abstract

Cellular edge features have been widely studied in recent years due to the interest in cell motility in normal and cancerous cells. Cues from cell interactions with the extracellular environment lead to overall cytoskeletal remodeling and consequently shape changes. The factors that determine the shape of a cell include actin polymerization, myosin II contraction and focal contacts (FCs). Actin based structures like lamellipodia (broad protrusions), ruffles (vertical protrusions) and filopodia (elongated, tapering protrusions) are the major protrusion types described in the scientific literature. However, their subjective classification is a problem when trying to understand mechanisms of protrusion formation in different cell types. The present study makes use of a previously determined computerized morphometric classification technique to distinguish features other than lamellipodia in 1000W rat tracheal epithelial cells. Some of the features identified through previous studies were factor 4, factor 5, factor 7 and factor 16. The aim of the present study was to explore the role of some of the structural components leading to the formation of a protrusion, particularly to a feature defined as factor 7. Integrated morphometry analysis with MetaMorph software was used to study the FC characteristics, while a geographical information system (GIS) software was used to study the FC orientation with respect to a protrusive feature. Small FCs favored factor 7 feature formation. High factor 7 values were observed when the class of FCs without actin became narrower. Paradoxically, where anchored actin cables on FCs showed a positive correlation with factor 7, the inner radius and width of the FC were the only elevated variables. The results suggested that certain populations of FCs with actin, which were narrow but varied in orientation, are important for factor 7 feature formation. It was observed that the most robust FCs with actin association were related to factor 5 values. The values of both factors 4 and 5 tended to increase with greater elongation of the FCs. FCs without actin cables affected the values of factor 4 and 5 to approximately the same extent. Factor 16 showed no correlations with any dimensioned or shape variable. Actin polymerization and myosin dependence in relationship to protrusions were studied with the aid of cytochalasins and myosin inhibitors, respectively. Cytochalasins inhibited ruffles and stress fibers but surprisingly enhanced factor 7 protrusions. Blebbistatin, SPC 16524, and H1152p, a Rho-kinase (ROCK) inhibitor, showed no ability to inhibit factor 7 features suggesting that myosin II was inessential to the features. The current study suggests that factor 7 feature resembles a pseudopodial feature enhanced by a Rho inhibitory p160/ROCK/p38 mitogen activated protein kinase signal transduction pathway in breast cancer invasiveness. Four models consistent with results were proposed for factor 7 formation. The activity of a minus-end directed motor was proposed in models II, III and IV, and no myosin function at all in model I. All the models ruled out myosin II activity, since the involvement of myosin II would require a contraction of stress fibers, a situation unlikely to generate a protrusion. Hence, the involvement of a minus-end directed motor is more probable. However, further data is required to rigorously test models II, III and IV. Since, model I, on the other hand was proposed to have no myosin activity, it still holds ground. The present study has enabled the identification of features that might aid as a tool in distinguishing normal and cancer cell populations. Disagreement with some aspects of protrusion formation suggests that not all protrusions are formed in the same manner and are under differential regulation. The study encourages the need to abandon subjective classifications of cellular edge features. There is certainly a...