Mechanobiology June 26th - June 2nd 2016

Mechanobiology: June 26th  - June 2nd 2016

The role of regulatory domain interactions in UNC-43 CaMKII localization and trafficking
Tohru Umemura, Paris Rapp, Christopher Rongo
  1. Fig. 1.

    The Caenorhabditis elegans CaMKII ortholog UNC-43 is localized to neurites. (A,B) UNC-43::CFP and (C,D) GLR-1::YFP can be detected in perinuclear structures in neuronal cell bodies (A,C) and clusters within the neurites of the ventral nerve cord (B,D). Merged images for A-D indicate that UNC-43 and GLR-1 colocalize (arrowheads) at perinuclear structures (E), and at a fraction of neurite clusters (F). Arrow in E indicates a unipolar neurite, partially out of the focal plane, extending from the cell body. Gray-scale images of UNC-43::GFP (G) and UNC-43(T286D)::GFP (H) indicate that the T286D change increases the amount of UNC-43 found diffusely distributed throughout the neurites. (I) Real-time PCR measurements of unc-43::gfp mRNA for nematodes expressing either one or two copies of an unc-43::gfp integrated transgene. Values are normalized to real-time PCR measurements of a dlg-1 control transcript. A significant difference in the level of expression was observed, **P<0.001 using Student's t-test (n=4), between the two groups. Fluorescence quantification measuring the mean fluorescent intensity values for neuron cell bodies (J) or neurites expressing either one copy or two copies of an unc-43::gfp transgene integrated into the genome (K). Fluorescence quantification measuring the mean fluorescent intensity values for neuron cell bodies (L), neurites including clustered and unlocalized fluorescence (M) and unlocalized fluorescence alone (N) (i.e., diffuse fluorescence defined by a threshold within two standard deviation values of the mean outside of the clustered signal) for nematodes expressing either UNC-43::GFP or UNC-43(T286D)::GFP. (O) The number of clusters (clusters defined as above a threshold of two standard deviation values of the mean outside of the clustered signal) per 10 μm length of neurite. (P) Frequency distribution plots of the mean fluorescence values of UNC-43::GFP clusters (straight black line) or UNC-43(T286D)::GFP clusters (dashed red line). A.U., arbitrary fluorescence units. Error bars indicate s.e.m. Significant differences *P<0.01, ***P<0.0001 were observed using the Student's t-test between the indicated test groups and the WT or single copy control group. n=10-25 animals per genotype. Bar 5 μm (A-F); 10 μm (G-H).

  2. Fig. 2.

    Amino acid interactions between the catalytic domain and the autoinhibitory domain regulate UNC-43 calcium-independent activity. (A) Amino acid sequence alignments between rat CaMKI, rat CaMKII and UNC-43 for three key domains: the active site core of the catalytic domain, the C-terminal lobe of the catalytic domain and the autoinhibitory domain. Residues changed in this study are indicated using rat CaMKII numbering. The CaMKII autophosphorylation site, calmodulin (CaM) binding site, and the pseudosubstrate domain are delimited by gray lines below the sequence. The secondary structure elements described for the CaMKI crystal structure are indicated above the lines, using gray boxes for α helices and white arrows for β strands (Goldberg et al., 1996). (B) A schematic model for CaMKII and UNC-43 regulatory amino acid interactions between the catalytic domain (the gray box) and the autoinhibitory domain (the black line and gray cylinder). Electrostatic interactions between amino acid side chains are indicated by a dotted line. Hydrophobic pockets created within the catalytic domain are indicated by the white ovals.

  3. Fig. 3.

    Autoinhibitory domain interactions regulate UNC-43 abundance in the neurites. Cell bodies and neurites expressing (A,B) wild-type UNC-43::GFP, (C,D) UNC-43(D238R)::GFP, (E,F) UNC-43(R283E)::GFP and (G,H) UNC-43(D238R, R283E)::GFP. Mutations in D238 result in the failure of UNC-43 to accumulate throughout the unlocalized neurite pool (D), whereas mutations in R283 (F) result in the overabundance of UNC-43 in this pool. The phenotype of the two mutations combined (H) is similar to that of D238R alone (D). (I,J) Fluorescence quantification measuring the mean fluorescent intensity values for neuron cell bodies (I) and neurites (J) expressing the indicated UNC-43::GFP construct. (K) The number of clusters (clusters defined as above a threshold of two standard deviation values of the mean outside of the clustered signal) per 10 μm length of neurite. WT indicates the wild-type UNC-43 protein, and an amino acid substitution number identifies each mutant form of UNC-43 tested. The catalytic domain, identified as UNC-43(1-270), and the subunit association domain, identified as UNC-43(271-520) were also tested. A.U., arbitrary units. Error bars indicate s.e.m. Significant differences were observed **P<0.01, ***P<0.001 compared to levels in the WT groups using one-way ANOVA followed by Dunnett's t-test comparisons. n=10-25 for each genotype. Bar, 5 μm.

  4. Fig. 4.

    UNC-43 calcium-dependent kinase activity. (A) S2 cells were transfected with the indicated cDNAs, then lysed and immunoblotted using anti-V5 antibodies to detect the recombinant proteins. (B) S2 cell lysates made from the indicated transfected cDNAs (squares for rat CaMKII and filled triangles for UNC-43) were tested for their ability to phosphorylate AC-2 in the presence of varying concentrations of calcium. The data were normalized to the maximal activity. (C) AC-2 kinase activities for S2 cell lysates from cells expressing the indicated cDNAs. White bars indicate the activity in the presence of calcium, whereas gray bars indicate the calcium-independent activity in the presence of EGTA. Error bars indicate the s.e.m. Significant differences in kinase activity were found *P<0.05 and ***P<0.001 by one-way ANOVA followed by Bonferoni comparisons between the same transgenes (calcium versus EGTA). n=3 or more trials. (D) The calcium-independent activity (AC-2 kinase activity in the presence of EGTA, plotted as a percentage of the activity in the presence of calcium) of S2 lysates expressing the indicated form of UNC-43. Error bars indicate s.e.m. Significant differences in activity were observed *P<0.05 and ***P<0.001, compared to levels in the WT group by one-way ANOVA followed by Bonferoni comparisons. n=3 or more trials.

  5. Fig. 5.

    UNC-43(K148E) and UNC-43(D238R) fail to rescue unc-43 mutants for GLR-1 trafficking. (A-G) GLR-1::GFP in neurons cell bodies and (H-N) SNB-1::GFP in neurites were examined in unc-43 mutants that also express either (A,H) no transgene, (B,I) a wild-type unc-43 cDNA, or unc-43 cDNA with the (C,J) K148E, (D,K) D238R, (E,L) H282K, (F,M) R283E or (G,N) T286D mutations. GLR-1::GFP is found in perinuclear puncta (arrowheads), and accumulates to high levels in these puncta in unc-43 mutants carrying no transgene (A), or expressing UNC-43(K148E) (B) or expressing UNC-43(D238R) (C). (O) Fluorescence quantification measuring the mean fluorescent intensity values for neuronal cell bodies expressing GLR-1::GFP and the indicated UNC-43 transgene. (P) SNB-1::GFP cluster number in neurites expressing the indicated UNC-43 construct. Significant differences in fluorescence intensity were observed *P<0.05, **P<0.01 and ***P<0.001 when compared to levels in the `None' group (no transgenic kinase) by one-way ANOVA followed by Bonferoni comparisons. The bar indicates a Bonferoni comparison between R283E and WT groups. n=10-30 animals per genotype.

  6. Fig. 6.

    UNC-2 regulates UNC-43 abundance in neurites. (A,C) Cell bodies and (B,D) neurites expressing wild-type UNC-43::GFP in (A,B) wild-type animals, or (C,D) in unc-2(e55) animals. Mutations in unc-2 result in the failure of UNC-43 to accumulate throughout the unlocalized neurite pool (D). Fluorescence quantification measuring the mean fluorescent intensity values for (E) neuron cell bodies and (F) neurites expressing UNC-43::GFP in either wild-type (WT) or unc-2(e55) mutant animals. (G) The number of clusters (clusters defined as above a threshold of two standard deviation values of the mean outside of the clustered signal) per 10 μm length of neurite for the indicated genotypes. A.U., arbitrary units. Error bars indicate s.e.m. Highly significant differences (***P<0.0001) in cluster number and neurite levels were observed in unc-2 mutants compared to levels in the WT control group using the Student's t-test. n=8 animals for each genotype. Bar, 5 μm.

  7. Fig. 7.

    Model for the amino acid interactions that regulate UNC-43 localization. A schematic model for UNC-43 regulatory amino acid interactions between the catalytic domain (the gray box) and the autoinhibitory domain (the black line and gray cylinder). Electrostatic interactions between amino acid side chains are indicated by a dotted line. Hydrophobic pockets created within the catalytic domain are indicated by the white ovals. A hypothetical factor that transports UNC-43 to the neurites is indicated by the white cylinder. Negatively-charged side chains are indicated by black circles, whereas positively-charged side chains are indicated by white circles. The black star in B indicates the addition of a phosphate at T286. (A) In the absence of activity, UNC-43 is kept quiescent by amino acid interactions between the catalytic domain and the autoinhibitory domain. (B) Upon calcium activation, calmodulin (not shown) binds and displaces the autoinhibitory domain, allowing for autophosphorylation at T286. The residues K148 and D238 are made available to bind to outside factors that facilitate the translocation of UNC-43 to neurites. (C) The T286D mutation mimics phosphorylation, even in the absence of calcium stimulation, by introducing charge at T286, displacing the autoinhibitory domain from the hydrophobic pocket along the catalytic domain. This constitutively exposes K148 and D238 for binding to outside factors, resulting in the over-transport of UNC-43 to the neurites. (D) The D238R mutation destabilizes an electrostatic interaction required for the autoinhibitory domain to bind to the catalytic domain, thereby resulting in constitutive kinase activity. The mutation also destabilizes a separate electrostatic interaction required for the catalytic domain to interact with the transport factors that facilitate UNC-43 localization to the neurites.