Project Mentor

Dr. Mallika Ghosh
Center for Vascular Biology

Undergraduate Research Opportunity Description

Project Description	Tunneling Nanotubes (TNTs) are specialized actin or microtubule-based membrane encased cell protrusions that hovering above the substratum, connecting two distant cells via one continuous structure for efficient cell-cell communication, transport of signaling molecules and cell organelles. Recent studies have reported TNT formation in a wide array of cells including neuronal, endothelial, as well as stem cells and immune cells. Cellular stress, apoptosis and inflammation are common inducers of TNT formation for spread of disease. CD13, an adhesion molecule mediating cell-cell and cell-ECM adhesion, has been shown to regulate internalization of receptors to control downstream signaling. Studies from our laboratory have implicated a critical role for CD13 in regulating actin cytoskeletal organization and receptor recycling as well. Given CD13’s function in endocytosis and actin cytoskeletal organization, we chose to explore CD13’s role in the formation and function of TNTs. Immunofluorescence and confocal microscopic analysis, revealed that cellular stress due to serum starvation induced formation of nanotubes in endothelial cells and macrophages. The majority of these TNTs were actin positive and carried early and recycling endosomes. Interestingly, serum starvation strongly induced CD13-positive TNT formation, while cells lacking CD13 showed significantly fewer nanotubes, suggesting that CD13 may have a critical role in the formation of these unique structures. Importantly, CD13 has been shown to be highly expressed in stem cells and early progenitor cells. Some recent studies have indicated that endothelial progenitor cells and mesenchymal stem cells (MSCs) have the ability to rescue damaged renal tubular cells by transferring newly generated functional mitochondria perhaps via nanotubes. We therefore propose to explore the functional role of CD13 in mediating nanotube formation and exchange of cargo between renal stem cells or MSCs and endothelial progenitor cells necessary to repair a damaged kidney in a murine model of unilateral ureteral obstruction. Since TNTs have been implicated in several physiological and pathological processes, understanding the underlying mechanism of TNTs formation and function may have great potential to contribute to developing novel therapeutic approaches in health and disease.
Project Direction	Expand and develop knowledge in the field of cell biology with specific focus in understanding microglial biology and Alzheimer’s disease. In our laboratory, we study the role of CD13 in receptor-mediated endocytosis and recycling, cell cytoskeleton organization and Amyloid beta induced tunneling nanotube (TNT) formation and function. TNTs are membrane bound protrusions that form between two distant cells sharing a continuous membrane but not attached to the substratum. We will explore the role of CD13 in the formation and function of Tunneling Nanotubes (TNTs) in innate immune cells of the brain such as microglia. We will perform the following experiments- i. Culture Microglia cell line (mouse and human) ii. Stain for microglia specific markers and CD13 in microglia cell lines and analyze by flow cytometry and immunohistochemistry and microscopy iii. Induce TNT formation and characterize the TNT/ microglia processes based on different expression of TNT markers and analyze by flow cytometry, immunohistochemistry and microscopy iv. Treat with labeled Amyloid beta and monitor transfer along the nanotube with sub cellular organelle markers v. Isolate primary brain microglia from prenatal and adult mouse and explore role of CD13 in structure and function of TNTs/processes vi. Characterize resident and activated mouse microglia and explore role of CD13 in structure and function of TNTs/processesLEARNING ACTIVITIESa) Perform assigned readings of scientific papers on previous research on Alzheimer’s disease, Microglial biology, Tunneling Nanotubes and CD13 b) Observe procedures performed by lab members as well as perform experimental procedures independently c) Observe procedures shown by mentor as well as perform tasks under supervision. d) Research new ideas and techniques in TNT field e) Have constant communication and feedback about procedures and techniques f) Follow lab protocols and if necessary develop new protocols as required g) Develop skill in tissue culture, Primary microglia isolation, Immunohistochemistry, Microscopy
Mentorship and Supervision	METHOD OF EVALUATION 1. Commitment to the Project- Attendance record and punctuality 2. Reflection Journal & Laboratory Notebook entry- Experimental skills, performance, experimental design, data interpretation and analysis 3. Presentations at weekly Lab Meetings
Student Qualifications	Specific skills: Cell culture, Immunostaining Coursework: Cell biology Academic interest: Biomedical science
Summer Schedule Options	Research Dates: May 28 to August 2, 2019 Schedule: M-F, 9am-5pm
Project Continuation	Fall 2019, Spring 2020
Academic Year Time Commitment	9 hours/week
Possible Thesis Project	Yes

Application

Submit an online application for this research opportunity at https://quest.uconn.edu/prog/HRP19-6/. The application deadline is Monday, February 4, 2019.

This application requires a cover letter, a resume or CV, an unofficial transcript, a brief statement of research interests, a brief statement of career interests, contact information for references, and letters of recommendation.