Title

Cellular Architecture and Cytoskeletal Structures Involved in Cell Haptotaxis

Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Carol Heckman, PhD

Second Advisor

Howard Cromwell, PhD (Committee Member)

Third Advisor

Carmen Fioravanti, PhD (Committee Member)

Fourth Advisor

Michael Geusz, PhD (Committee Member)

Fifth Advisor

Wendell Griffith, PhD (Committee Member)

Abstract

Filopodia play a sensory role in directing motility during embryonic development and axon pathfinding. They also show a low prevalence in cancer cells. Here, I determined whether cultured cells from a rat tracheal epithelial line used filopodia to sense adhesive gradients. Cells exhibited haptotaxis (movement toward the more adhesive surface) when plated on tantalum (Ta) and platinum (Pt) metallic gradients. The gradients were created on glass, and high (H), middle (M), and low (L) positions defined along the gradient. Cell counts in randomly selected fields confirmed that the cells recognized the gradient. To determine whether the prevalence of protrusions differed at the H, M, and L locations, the values of latent factors 4 (filopodia), 5, and 7 were determined. Factor 4 values were high at H and significantly lower at M and L (p<0.05). Cells' ability to form larger protrusions, represented by factors 5 (lamellar distribution) and 7 (nascent neurites), was unaltered across the Ta gradient. The directional cues also appeared to be interpreted within a cell, as shown by analyzing the cells' top (T) side, i.e. the side oriented toward H location, from the bottom (B) side. Factor 4 values at H-T significantly exceed those at M-B and L-T. Trend analysis confirmed a decrease in factor 4 over the gradient with significance of P<0.001 (Ta) and P<0.023 (Pt). On Pt only, factor 5 increased (P<0.0001) as the metal content of the substrate declined.

Cdc42 (cell division cycle 42) plays a crucial role in establishing polarity, and is also involved in filopodia formation, cell motility, and directional migration. Since there is some loss of polarity in preneoplastic lesions, the role of Cdc42 in filopodia-mediated sensing was of interest. To determine whether Cdc42 was implicated in reconstruction of cellular architecture and rearrangement of cytoskeletal structures, I tested the role of Cdc42 effectors in gradient sensing. While most effectors bind to Cdc42 at multiple regions, there is usually a short linear stretch of residues that is critical for binding. Peptides representing such stretches of Cdc42 were designed to model its surface and thereby inhibit effector-Cdc42 binding. Using filopodia trend analysis to determine the effect of each peptide, I found that ACK, IQGAP, and Par6 were essential for directional pointing of filopodia. A sequence blocking PKCε interaction with its docking site also prevented pointing activity. Although WASP binds directly to Cdc42, the peptide designed to block this interaction did not affect directional pointing of the filopodia. Introduction of the peptide against PAK gave a paradoxical result. Directional identification mechanisms were intact but the direction of filopodia pointing was reversed.

I also studied the relationship between filopodia and ruffles on any given cell, in order to understand the complex processes of cell motility and directional persistence. Ruffle frequency and filopodia were inversely related, and this relationship was independent of the location on the gradient or the peptide introduced into the cells. On tantalum, sites at HT, MT, and LT and on platinum sites at HT, MT, and both LT and LB showed ruffling interacting with filopodia, with a level of significance P<0.025. Despite the tendency for ruffling activity to increase toward the less adhesive end of the gradient, none of the ruffling variables showed a statistically significant trend. However, the data suggest that cells make ruffles at the expense of filopodia regardless of substrate to which they are attached. This inverse relationship was stronger at the top of the cell than at the bottom. Thus it can be confirmed that ruffles and filopodia are inversely related, but ruffling is unlikely to be a mechanism of gradient sensing.