The Lim domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans

Broday L. et al.

June issue of JCB

Ruttenberg Cancer Center (NY)

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Back to the paper…

Brief overview of C. elegans muscle

Role of UNC-95 in sarcomere assembly

Role of RNF-5 in localization of UNC-95

Muscle Structure (C. elegans II)

Why UNC-95?

LIM domain proteins have recently been shown to aid in assembly of these structures

UNC-97 is a LIM domain protein and has a central role

UNC-95 is a LIM domain protein that was previously uncharacterized

unc-95 Mutants

Zengal and Epstein (1980) found 2 mutantsVery slow to paralyzedLack of striations and disorganized thick and

thin filaments

Mutant Phenotype

Long arrows indicate cell/cell boundaries, short arrows indicate dense bodies

Disorganized thin filaments

Disorganized thick filaments

Disorganized dense bodies

Disorganized dense bodies

EM of unc-95 animals

Irregular dense bodies

Random dense body spacing

Barely recognizable M-line

Disorganized thin filaments

Disorganized thick filaments

No recognizable I-line

Characterization of unc-95 (su33)

Y105E8.6 found associated with RNF-5 in a Y2H screen

Hypothesized to be unc-95 Y105E8.6 was sequenced in su33 mutant

and found to have a CAG TAG mutation causing a truncated protein without LIM domain

Rescue with functional fusion

GFP translational fusion with standard 2.5kb upstream (promoter) created

Worms were injected with construct and assayed

RT-PCR showed that the mutant gene was in fact transcribed

Rescue with functional fusionA,B,C: Rescue with translational fusion

D,E: Protein structure

F: RT-PCR of mutant and wild type show comparable transcription

G,H,I: RNAi with Y105E8A.6 construct

Role of UNC-95 during embryogenesis

A-F: anti-UNC-52/perlecan staining showing wild type phenotype in mutant

G-N: anti-PAT-3/ integrin staining shows wild type phenotype until post-hatching

O-T: anti DEB-1/vinculin staining is diffuse in all mutant stages

Conclusions from this data

UNC-95 not required for localization of UNC-52 perlecan in basement membrane

Recruitment of -integrin to basal sarcolemma not dependant on UNC-95

UNC-95 is required for recruitment of vinculin

Analysis of unc-95 localization

A-I: Expression is seen throughout muscle cells but especially in cellular attachment sites as indicated by the various arrows

J-L: Expression of truncated unc-95 translational fusion shows low overall expression and no expression at cellular attachment sites

Role of RNF-5

Colocalizes with UNC-95 in dense bodies

Regulates levels of UNC-95

Regulates UNC-95 subcellular location

Colocalization of RNF-5 and UNC-95

A,B: anti-RNF-5 and anti-DEB-1-vinculin

C,D: same as A,B but with RNAi

E: rnf-5 mutant stained with anti-DEB-1-vinculin

F: unc-95 anti RNF-5

G: localization of RNF-5 in dense bodiesH: colocalization of RNF-5 and UNC-95 in yellow

RNF regulation of UNC-95

A: UNC-95::GFP

A: UNC-95::GFP with RNF-5 overexpressed with a heat shock promoter

A: UNC-95::GFP with overexpression of a truncated form of RNF-5 (no RING finger domain)

Conclusion: an intact RING finger domain is required for proper regulation of UNC-95

RNF-5 RNAi

RNAi was used to deplete the levels of RNF-5 and an increase in GFP expression from UNC-95::GFP is seen in B and C

In heterozygous rnf-5 mutants, a similar increase in GFP expression is seen in (D and E)

Summary

Y105E8.6 is unc-95 UNC-95 is required for proper recruitment

of vinculin for initial assembly of muscle attachment sites

UNC-95 is localized primarily to muscle attachment sites

RNF-5 colocalizes with UNC-95 and regulates its location and levels

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