Dissection of CD2AP functions in epithelial cells

I have investigated a molecular mechanism that determines the shape of epithelial cells. Epithelial cells are characterized by extensive cell-cell contacts along lateral membranes that hold the cells together in cohesive sheets and by a pronounced apical-basal polarity in which the apical domain faces a lumen and the basal domain contacts the basement membrane of the extracellular matrix. Therefore, all epithelial cells must build three biochemically and functionally distinct plasma membrane domains: apical, basal, and lateral. The relative proportions of these three domains vary between epithelial cell types and are coupled to epithelial cell functions. However, surprisingly little is known as to what controls membrane proportions. I discovered a critical role for a molecular scaffolding protein known as CD2AP in controlling the height of the lateral membrane and the area of the apical and basal membrane domains. Depleting CD2AP resulted in a dramatic shortening of the lateral membrane but an expansion of the apical and basal membranes such that the cells spread extensively but become very thin as they convert from a cuboidal to squamous epithelial morphology. These changes in cell morphology phenocopy those generated by inhibiting PI3 Kinase. I confirmed that blocking PI3 Kinase activity led to a decrease in cell height. I therefore tested if there was a molecular, mechanistic connection between CD2AP and PI3 Kinase in controlling epithelial cell height. Immunofluorescence staining showed that CD2AP co-localized with PI3K p85 while biochemical analysis showed that CD2AP binds directly to p85 in vitro. Depleting CD2AP in cells showed that it is necessary for localizing PI3K to cell-cell boundaries and for PIP3 accumulation along lateral membranes. CD2AP is also necessary for actin assembly in epithelial cells, and I demonstrated that perturbing actin assembly with drugs also leads to an expansion of apical and basal domains that comes at the expense of lateral membrane height. A structure-function analysis of CD2AP showed that the targeting the SH3 domains (1-329 a.a.) of CD2AP, which bind to PI3 Kinase, to the membrane was sufficient to restore p85 and p110 membrane levels. Tethering either CD2AP 1-329 a.a. or PI3K p110 itself to membranes rescued PIP3 accumulation, F-actin accumulation, and membrane proportions in CD2AP-knockdown MDCK cells. Thus, CD2AP recruits PI3K to regulate cell height. In addition to this work, I demonstrated that CD2AP is itself an F-actin binding protein that alters actin filament polymerization dynamics. In addition, I found that CD2AP binds directly to a known actin elongation factor known as EVL. Interestingly, earlier work suggested that EVL itself might also be necessary for building up lateral membranes and constraining cell area. These results begin to suggest the possibility of a positive feedback loop in which CD2AP, perhaps in conjunction with EVL, couple actin filaments to PI3 Kinase signaling to determine epithelial cell height. These results should provide new insights into squamous to cuboidal to columnar epithelial transitions seen in complex epithelial tissues in vivo.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Cell and Developmental Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2020

Author dc:creator

• Wang, Yuou

Contributors dc:contributor

• Brieher, William M
• Chen, Jie
• Ceman, Stephanie S
• Leckband, Deborah E

• CD2AP
• PI3K
• cell shape
• actin

Statement dc:rights

• Copyright 2020 William M. Brieher

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/108672

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/108672