Transcript unc-95 - Department of Zoology, UBC

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
mutants
 Very
slow to paralyzed
 Lack of striations and disorganized thick and
thin filaments
Mutant Phenotype
Disorganized
thin filaments
Disorganized
thick filaments
Disorganized
dense bodies
Disorganized
dense bodies
Long arrows indicate cell/cell boundaries, short arrows indicate
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 fusion
A,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 DEB1/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 bodies
H: 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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