Alpha Omega Alpha Honor Medical Society

Characterizing the Role of IQGAP1 in Leukocyte Transmigration

Investigator: Parthana Dalal

Mentors: William A. Muller, MD, PhD

Introduction:
Inflammation is an essential, physiologic process that is normally well adapted to protect against infection and heal wounds. However, dysregulated inflammation can cause significant damage to host tissues resulting in conditions such as atherosclerosis. This project seeks to examine the cellular mechanisms of how leukocytes leave the circulation and move into damaged tissues. In particular understanding the role of endothelial cell IQ-domain GTPase- activating protein 1 (IQGAP1) can help define the key cell signaling events involved in permitting leukocyte transendothelial migration (TEM). IQGAP1 is a large scaffolding protein known to integrate signals from diverse pathways to regulate outcomes like cytoskeletal remodeling in other processes but its role in leukocyte transmigration has not been well-studied. Using fluorescently tagged IQGAP1 truncation constructs we were able to show that the actin- binding domain and the IQ motif domain are important for allowing leukocyte passage. Building on these findings we are now testing point mutations within these regions in an attempt to isolate IQGAP1 interacting factors involved in the cellular changes of TEM.

Methods:
The transendothelial migration assay was performed by isolating peripheral blood mononuclear cells (PBMCs) and neutrophils through density gradient centrifugation. These cells were then added to the immortalized human umbilical vein endothelial cell (iHUVEC) monolayers to allow TEM to ensue. After one hour the endothelial cells and leukocytes were fixed and stained. TEM is analyzed by manually counting cells and noting the position of the leukocytes as above or below the endothelial monolayer.

Results:
We confirmed our preliminary findings that IQGAP1 is essential for TEM and optimized the conditions for this viral knockdown. In the process we had to reengineer a few constructs to optimize the knockdown of the endogenous protein. Figure 1 shows that IQGAP1 knockdown significantly impairs the leukocyte ability to cross endothelial monolayers and this is comparable to blocking PECAM (a known control for inhibiting TEM). Figure 2 shows point mutant constructs of IQGAP1 in regions key for its function in TEM (IQ motif domain). We validated that these are optimal for future use. These constructs are expressed in adenovirus vectors and are in the process of being tested in vitro. In preliminary data it seems as though construct IQGAP1-ID GFP may impair TEM function and this work has already served as a platform for the next phase of investigation for the project in the lab.

Discussion:
Understanding the molecular signaling involved in coordinating inflammation and allowing leukocyte passage into damaged tissues can help provide novel directions for research in a wide variety of diseases driven by host immune dysfunction. By establishing that IQGAP1 is important for TEM and that specifically the IQ motif domain is critical for that function we can begin to identify potential interacting candidates. We currently hypothesize that the IQ motif domain interacts with calmodulin to integrate calcium signaling with cytoskeletal change required in endothelial cells to allow leukocyte passage. Calmodulin has been shown to interact with IQGAP1 and specifically with isoleucine residue mutated in Construct 2 (IQGAP1-ID- GFP). We hope that by further studying this point mutant construct we can isolate this interaction and establish whether it is important in the TEM process. We are also in the process of testing IQGAP1 knockout mice and using microscopy to visualize in vivo defects in leukocyte transmigration.

Last modified: 2/03/2017

Updated on February 3, 2017.


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