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First published online October 27, 2005
doi: 10.1242/10.1242/jcs.02611

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The JIL-1 kinase interacts with lamin Dm₀ and regulates nuclear lamina morphology of Drosophila nurse cells

Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA

View larger version (47K): [in a new window]   Fig. 1. Lamin Dm0 interacts with JIL-1 in pull-down assays. (A) S2 cell lysate incubated with JIL-1-NTD (aa 1-211) or JIL-1-CTD (aa 927-1207) GST-fusion protein constructs or with a beads-only control was pelleted with glutathione-agarose beads and the interacting protein(s) fractionated by SDS-PAGE, western blotted, and probed with the lamin mAb HL1203. Unincubated S2 cell lysate was included as a control (lane 1). Only the JIL-1-CTD construct was able to pull down the 76 kDa lamin protein (lane 4) also detected in the cell lysate, whereas no interaction was observed with the GST-only control (lane 2) or the JIL-1-NTD construct (lane 3). (B) Immunoblot of the input GST-fusion proteins (JIL-1-NTD and JIL-1-CTD) used for the pull-down experiments in A detected with the anti-GST mAb 8C7. (C) S2 cell lysate incubated with a lamin-GST fusion construct (aa 260-622) or a GST-only control or with beads-only was pelleted with glutathione-agarose beads and the interacting protein(s) fractionated by SDS-PAGE, western blotted, and probed with affinity purified JIL-1 antiserum. Unincubated S2 cell lysate was included as a control (lane 1). The lamin-GST fusion protein construct was able to pull down the 160 kDa JIL-1 protein (lane 4) also detected in the cell lysate while no interaction was observed with the GST-only control (lane 3) or with the beads-only (lane 2). (D) Immunoblot of the input GST-fusion protein (lamin-GST) and the GST control used for the pull-down experiments in C detected with the anti-GST mAb 8C7. The relative migration of molecular weight markers is indicated to the right in kDa.

View larger version (17K): [in a new window]   Fig. 2. JIL-1 and lamin Dm0 immunoprecipitation assays. (A) Immunoprecipitation (ip) of S2 cell lysate was performed using the lamin mAb HL1203 (lane 1) and the leech tractin control mAb 1H4 (lane 2) coupled to immunobeads or with immunobeads only (lane 3). The immunoprecipitations were analyzed by SDS-PAGE and western blotting using JIL-1 antiserum for detection. JIL-1 antiserum staining of S2 cell lysate is shown in lane 4. JIL-1 is detected in the lamin immunoprecipitation sample as a 160 kDa band (lane 1) but not in the control samples (lanes 2 and 3). (B) Immunoprecipitation (ip) of S2 cell lysate was performed using JIL-1 antiserum (lane 1) as well as a pre-immune serum control (lane 2) coupled to immunobeads or with immunobeads only (lane 3). The immunoprecipitations were analyzed by SDS-PAGE and western blotting using lamin mAb HL1203 for detection. Lamin mAb HL1203 staining of S2 cell lysate is shown in lane 4. Lamin is detected in the JIL-1 antiserum immunoprecipitation sample as a 76 kDa band (lane 1) but not in the pre-immune or beads-only control samples (lanes 2 and 3). The relative migration of molecular weight markers is indicated to the right in kDa.

View larger version (31K): [in a new window]   Fig. 3. Mapping of the JIL-1 interaction domain with lamin Dm0. (A) The truncated COOH-terminal JIL-1 GST-fusion protein constructs used for domain mapping. The JIL-1 COOH-terminal domain can be divided into predominantly acidic and basic regions, with the basic region containing a predicted globular domain. (B) S2 cell lysate was incubated with the various truncated JIL-1 GST-fusion protein constructs shown in A or with a beads-only control and pelleted with glutathione-agarose beads. Interacting protein(s) were fractionated by SDS-PAGE, western blotted, and probed with the lamin mAb HL1203. Unincubated S2 cell lysate was included as a control (lane 6). The CTD, CTD-B and CTD-G were able to pull down the 76 kDa lamin protein (lanes 1, 3 and 4) also detected in the cell lysate (lane 6), whereas no interaction was observed with the CTD-A construct (lane 2) or with the beads-only control (lane 5). This defined the globular domain in the basic region as sufficient for mediating interactions with lamin Dm0. (C) Immunoblot of the input GST-fusion proteins used for the pull-down experiments in (B) detected with the anti-GST mAb 8C7. The relative migration of molecular weight markers is indicated to the right in kDa.

View larger version (28K): [in a new window]   Fig. 4. Mapping of the lamin Dm0 interaction domain with JIL-1. (A) The truncated COOH-terminal lamin Dm0 GST-fusion protein constructs used for domain mapping. The Ig-like fold of the lamin Dm0 tail domain (white) is shown in black. Some of the constructs also contained parts of the rod domain (shown in gray). (B) S2 cell lysate was incubated with the various truncated lamin GST-fusion protein constructs shown in A or with a beads-only control and pelleted with glutathione-agarose beads. Interacting protein(s) were fractionated by SDS-PAGE, western blotted and probed with JIL-1 antiserum. Unincubated S2 cell lysate was included as a control (lane 6). The lamin-D1 and lamin-D3 constructs were able to pull down the 160 kDa JIL-1 protein (lanes 1 and 3) also detected in the cell lysate (lane 6), whereas no interaction was observed with the lamin-D2 and lamin-D4 constructs (lanes 2 and 4) or with the beads-only control (lane 5). This defined the lamin-D3 domain in the tail region as sufficient for mediating interactions with JIL-1. (C) Immunoblot of the input GST-fusion proteins used for the pull-down experiments in B detected with the anti-GST mAb 8C7. The relative migration of molecular weight markers is indicated to the right in kDa.

View larger version (141K): [in a new window]   Fig. 5. Egg chambers from JIL-1 mutant flies labeled with lamin Dm0 antibody. (A) Wild-type (WT) egg chamber labeled with lamin pAb R836. The location of follicle cells (f), nurse cells (n) and the oocyte (o) is indicated by arrows. (B) Egg chamber from a JIL-1z2/JIL-1h9 (z2/h9) mutant fly labeled with lamin pAb R836. The arrows indicate dispersed and mislocalized lamin Dm0 throughout the egg chamber. (C,D) Egg chambers from a JIL-1z2/JIL-1h9 (z2/h9) mutant fly labeled with lamin pAb R836. The arrows point to protrusions of the nuclear lamina of nurse cells near the oocyte. (E,F) Egg chambers from a JIL-1z2/JIL-1h9 (z2/h9) mutant fly double-labeled with lamin pAb R836 (green) and phalloidin (red). The protrusions from the nuclear lamina of the nurse cells extend through the ring canals. Bars, 25 µm in A-D and 10 µm in E-F.

View larger version (18K): [in a new window]   Fig. 6. Genetic interaction between JIL-13657 and Lam alleles. (A) Presence of a heterozygous Lam4643 allele increases the viability of JIL-13657 homozygous animals. Lam4643/CyO; JIL-13657/JIL-13657 males were mated with Sp/CyO; JIL-13657/JIL-13657 females. Homozygous JIL-13657 individuals that were also homozygous for the wild-type Lam allele (histogram to the right) eclosed at a rate only about half (0.44) that of JIL-13657 homozygotes that contained one copy of the loss-of-function Lam4643 allele (histogram to the left). The average ratios of adult flies of each genotype to total eclosed flies are from 13 independent matings. The difference in numbers observed for the two classes was statistically significant (P<0.0001, Student's two-tailed t-test). (B) The presence of a heterozygous LamA25 allele does not affect the viability of JIL-13657 homozygous animals. LamA25/CyO; JIL-13657/JIL-13657 males were mated with Sp/CyO; JIL-13657/JIL-13657 females. Homozygous JIL-13657 individuals that were also homozygous for the wild-type Lam allele (histogram to the right) eclosed at the same rate (0.97) as JIL-13657 homozygotes that contained one copy of the LamA25 allele (histogram to the left). The average ratios of adult flies of each genotype to total eclosed flies are from 21 independent matings. The difference in numbers observed for the two classes was not statistically significant (P>0.8, Student's two-tailed t-test).