Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Summary

Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H.

Abstract

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 ± 0.30 μm/sec in R256H cells compared to 1.60 ± 0.42 μm/sec in wild type cells, p < 0.005).

Introduction

Actin is the dominant protein comprising the cytoskeleton and participates in critical cellular processes including cell division, organelle movement, cell motility, contraction, and signaling. Over the past decade, disease-causing mutations in actin have been discovered in each of the six human actin isoforms leading to a range of disorders, from myopathies to coronary artery disease^1-7. The processes by which actin mutations lead to disease continue to be elucidated. The yeast model remains the gold standard to study the biochemical effects of mutations on actin function owing to the advantages of the single essential actin isoform, genetic tractability a....

Protocol

1. Cloning into the PB1996 Plasmid

1. Design and order DNA primers from an oligonucleotide manufacturing company that flank the target sequence and contain unique restriction sites in the selected parent plasmid. NOTE: In this case, primers were designed to amplify the 400 base pairs at the 5’ end of the Aip1 sequence. The XhoI restriction site was incorporated into the primer about 20 bp before the Aip1 sequence, and XmaI was included about 20 bp after the target Aip1 sequence. The plasmid used for cloning was PB1996, which contains a 3XGFP tag for fluorescence, a URA gene for yeast strain selection, and an ampicillin resistance gene for bacterial sele....

Results

A method to image the dynamics of cytoskeletal proteins in the cell is presented. The actin-binding protein, Aip1p, was tagged with GFP. The design for the plasmid encoding the tagged product is shown in Figure 1. The plasmid was then transformed into the yeast cells. Expression of the fluorescently tagged Aip1p allowed visualization of the protein behavior in the cell. Aip1p typically localizes to actin patches at sites of endocytosis²². To quantify Aip1p movement, more than 50 fluorescent Ai.......

Discussion

An effective strategy to visualize the dynamics of the cytoskeleton and the utility in investigations on pathogenic mutations has been described here. Advanced imaging modalities have created new opportunities to understand the intracellular movement of proteins near the cell membrane. Total internal reflection fluorescence microscopy (TIRF) is a sensitive technique for functional studies in living cells. TIRF uses an angled excitation laser that creates an evanescent field due to the difference in refractive indexes of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	rpi	9012-36-6
Bromophenol Blue	Amresco	115-39-9
BSA	NEB	B9001S
Change-IT Multiple Mutation Site Directed Mutagenesis Kit	USB Corporation	4166059
CutSmart Buffer	NEB	B7204S
DNA, single stranded from salmon testes	Sigma	9007-49-2
EDTA pH 7.4	Sigma	93302
Ethidium bromide	Invitrogen	15585-011	Warning! Harmful irritation
Fungal/Bacterial DNA Kit	Symo Research	D6005
HpaI	NEB	R0105S
Lithium acetate	AlfaAesar	6108-17-4
Low DNA Mass Ladder	Invitrogen	10068-013
NE Buffer #4	NEB	B7004S
Platinum PCR SuperMix High Fidelity	Invitrogen	12532-016
Miniprep Kit	Qiagen	27106	Any kit will work
Quick Ligation Kit	NEB	M2200S
Sodium azide	Sigma	26628-22-8
PBS	Invitrogen	10010-023
PEG	Amresco	25322-68-3
Tris Base Ultrapure	rpi	77-86-1
Wizard SV Gel and PCR Clean-Up System	Promega	1/6/2015
XhoI	NEB	R0146S
XmaI	NEB	R0180S
YPD media	LabExpress	3011
-URA Media	LabExpress	3010
PCR Machine	Invitrogen	4359659	Any PCR machine will work
TIRF Microscope	Olympus IX81
Hamamatsu ORCA-R camera	Hamamatsu