Document Type

Honors Capstone Project

Date of Submission

Spring 5-1-2013

Capstone Advisor

Vladimir Sirotkin

Honors Reader

Scott Erdman

Capstone Major

Biology

Capstone College

Arts and Science

Audio/Visual Component

no

Capstone Prize Winner

no

Won Capstone Funding

yes

Honors Categories

Sciences and Engineering

Subject Categories

Biology

Abstract

In yeast and other organisms, endocytosis is dependent upon Arp2/3 complex-mediated actin assembly into endocytic actin patches, a highly organized process involving signaling molecules and regulatory proteins. The goal of this study is to determine the roles of three Arp2/3 complex activators Pan1, Myo1, and Wsp1, which bind Arp2/3 complex via their Central-Acidic (CA) domains, in stimulating actin patch assembly in fission yeast. To assess the contribution of these proteins to actin assembly, we performed quantitative image analysis of actin patch dynamics by tracking the GFP-tagged actin binding protein fimbrin Fim1 in Pan1, Myo1 and Wsp1 mutants with deleted CA domains. In myo1ΔCA, like in the wild type cells, most patches internalized and patch dynamics was the same as in the wild type strain. The Pan1 mutant contains a deletion of the ACV region rather than just the CA region like Myo1 and Wsp1 due to the fact that the region is inverted in the Pan1 mutant. In pan1ΔACV, most patches internalized but accumulated 2.3 times more actin and at the faster rate than in the wild type cells. In wsp1ΔCA, the majority of the patches failed to internalize, indicating failed endocytosis, and the rate of patch assembly and the total actin accumulation in the patch were decreased compared to the wild type cells. Next, we used genetic crosses and tetrad analysis to determine which of the CA domains of the Arp2/3 complex activators are essential for cell viability. We observed that combinations of myo1ΔCA with pan1ΔACV and myo1ΔCA with wsp1ΔCA resulted in viable cells, while the combination of wsp1ΔCA and pan1ΔACV mutants was synthetically lethal. This suggests that activation of the Arp2/3 complex by Wsp1 alone or Pan1 alone but not Myo1 alone is minimally sufficient for cell viability. Imaging Fim1-mGFP in cells with viable combinations of ΔCA mutations revealed that both the myo1ΔCA pan1ΔACV cells and the myo1ΔCA wsp1ΔCA cells contained actin patches, indicating that activation of Arp2/3 complex by either Wsp1 or Pan1 alone suffices to promote actin patch assembly. However, given the patch internalization defect in wsp1ΔCA but not in pan1ΔACV or myo1ΔCA mutants, we concluded that Wsp1 is the primary Arp2/3 complex activator at the endocytic sites, minimally sufficient to support actin assembly, cell viability, and endocytic internalization.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Included in

Biology Commons

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.