20 - Subcellular Targeting and Analysis of Rac and CDC42 in MDCK Cells

Author Information

Tyler RectorFollow

Description/Abstract/Artist Statement

Mechanobiology is the study of how mechanical forces influence cellular function. Rho GTPases are signaling proteins that regulate multiple aspects of the internal cytoskeleton, cellular migration, morphology, and development of the cell. We hypothesize that optogenetic activation of the Rho GTPases, Rac and cdc42, will initiate the formation of lamellipodia and filopodia along the leading edge of a cell. These structures are dynamic, actin-rich protrusions that play critical roles in sensing the environment, enabling cell movement, and adapting cellular architecture. Optogenetic plasmid constructs enable the activation and recruitment of these Rac and cdc42 proteins. This project utilized the blue light-inducible plasmid ILID-CAAX paired with the ITSN-mCherry-sspB plasmid to test the localized activation of cdc42 on generating actin-rich protrusions like lamellipodia or filopodia. Pattern illumination allows for the selective illumination of specific regions of a biological sample. When paired with the outlined plasmid constructs, this approach allows for targeted subcellular activation. We have successfully transfected Madin-Darby Canine Kidney (MDCK) cells with the aforementioned plasmids. Upon global activation and recruitment of cdc42 utilizing the optogenetic plasmid constructs ILID-CAAX and ITSN-mCherry-sspB in MDCK cells, rapid aggregation of ITSN-sspB was observed, which rapidly dissipated within 25 secs of activation. Preliminary observations suggest that targeted movement occurs following optogenetic activation of cdc42, but further testing is required to confirm and characterize this effect. Our approach will enable us to understand the role Rho GTPases play in cellular adhesion, morphology, and motility.

Presenting Author Name/s

Tyler Rector

Faculty Advisor/Mentor

Venkat Maruthamuthu

Faculty Advisor/Mentor Department

Mechanical & Aerospace Engineering

College Affiliation

College of Engineering & Technology (Batten)

Presentation Type

Poster

Disciplines

Biomechanical Engineering | Biotechnology | Integrative Biology | Other Biochemistry, Biophysics, and Structural Biology

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20 - Subcellular Targeting and Analysis of Rac and CDC42 in MDCK Cells

Mechanobiology is the study of how mechanical forces influence cellular function. Rho GTPases are signaling proteins that regulate multiple aspects of the internal cytoskeleton, cellular migration, morphology, and development of the cell. We hypothesize that optogenetic activation of the Rho GTPases, Rac and cdc42, will initiate the formation of lamellipodia and filopodia along the leading edge of a cell. These structures are dynamic, actin-rich protrusions that play critical roles in sensing the environment, enabling cell movement, and adapting cellular architecture. Optogenetic plasmid constructs enable the activation and recruitment of these Rac and cdc42 proteins. This project utilized the blue light-inducible plasmid ILID-CAAX paired with the ITSN-mCherry-sspB plasmid to test the localized activation of cdc42 on generating actin-rich protrusions like lamellipodia or filopodia. Pattern illumination allows for the selective illumination of specific regions of a biological sample. When paired with the outlined plasmid constructs, this approach allows for targeted subcellular activation. We have successfully transfected Madin-Darby Canine Kidney (MDCK) cells with the aforementioned plasmids. Upon global activation and recruitment of cdc42 utilizing the optogenetic plasmid constructs ILID-CAAX and ITSN-mCherry-sspB in MDCK cells, rapid aggregation of ITSN-sspB was observed, which rapidly dissipated within 25 secs of activation. Preliminary observations suggest that targeted movement occurs following optogenetic activation of cdc42, but further testing is required to confirm and characterize this effect. Our approach will enable us to understand the role Rho GTPases play in cellular adhesion, morphology, and motility.