Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor.
Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.
University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States; Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.
Satoshi
S
Okada
The Ohio State University, Columbus, OH, United States.
Mid Eum
ME
Lee
University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States. Electronic address: ebi@mail.med.upenn.edu.
Erfei
E
Bi
The Ohio State University, Columbus, OH, United States. Electronic address: park.294@osu.edu.
Hay-Oak
HO
Park
D015374:Biosensing Techniques / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D061848:Optical Imaging / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D004718:Saccharomycetales / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D020764:cdc42 GTP-Binding Protein / Q000032:analysis* / Q000378:metabolism; D000097573:Red Fluorescent Protein
D005656:Fungal Proteins; D006160:Guanosine Triphosphate; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D020764:cdc42 GTP-Binding Protein; D000097573:Red Fluorescent Protein
CRIB; Cdc42; FRAP; GEF; GFP; GTPase