D-Titin: a giant protein with dual roles in chromosomes and muscles

Abstract

Previously, we reported that chromosomes contain a giant filamentous protein, which we identified as titin, a component of muscle sarcomeres. Here, we report the sequence of the entire titin gene in Drosophila melanogaster, D-Titin, and show that it encodes a two-megadalton protein with significant colinear homology to the NH(2)-terminal half of vertebrate titin. Mutations in D-Titin cause chromosome undercondensation, chromosome breakage, loss of diploidy, and premature sister chromatid separation. Additionally, D-Titin mutants have defects in myoblast fusion and muscle organization. The phenotypes of the D-Titin mutants suggest parallel roles for titin in both muscle and chromosome structure and elasticity, and provide new insight into chromosome structure.

Figure 1

Molecular characterization of the D-Titin gene. (A) Molecular map of the region in 62C containing D-Titin. A series of seven overlapping contigs span the entire D-Titin gene and link previously isolated D-Titin clones (KZ, NB, LG, JT) and four new D-Titin cDNAs (GH05716, CK340, CK55, LD28564). D-Titin spans from nucleotide (nt) 293742 (5′) to nt 182959 (3′) in Celera contig AE003473.1. The ORF in the LG clone was completely contained within the large contig, however; LG is a chimeric genomic clone that also includes noncoding sequences from cytological region 62B. The kettin sequence (Hakeda et al. 2000; Kolmerer et al. 2000) is entirely included within the D-Titin gene. Three P-element insertions in D-Titin, l(3)rL182, v(3)04860, and l(3)j1D7, are indicated, as well as the flanking DNA, isolated by plasmid rescue (PR4860 and PRj1D7). l(3)6265 is a P-element insertion used to clone flanking genomic DNA and map the proximal breakpoint of Df(3L)Aprt123. Df(3L)Aprt123 deletes the neighboring distal gene (mu2) but does not affect D-Titin function. (B) Protein expression profiles detected by an antibody against D-TITIN (α-KZ) (a–f) and an antibody against KET (Lakey et al. 1993) (g–l). Identical expression is detected in all the somatic (sm), visceral (vm), and pharyngeal muscles (ph) and their precursors during embryogenesis with both antibodies. (a and g) Stage 11 embryos, ventral view. (b and h) Stage 13 embryos, ventral view. (c and i) Stage 16 embryos, ventral view. (d and j) Stage 11 embryos, lateral view. (e and k) Stage 13 embryos, lateral view. (f and l) Stage 16 embryos, lateral view. All embryos are oriented with anterior to the left. For embryos shown in lateral view, dorsal is up. (C) D-Titin transcripts detected by whole-mount in situ hybridization. Genomic and cDNA clones mapping throughout the D-Titin gene (see gray lines in A) detected identical expression patterns throughout embryogenesis in all striated muscles and their precursors. Shown in a–d are embryos hybridized with the JT cDNA, which encodes a portion of the PEVK-2 domain. (a) ventral view of stage 13 embryo; (b) lateral view of stage 14 embryo; (c) ventral view of early stage 15 embryo; (d) lateral view of stage 16 embryo. (D) Domain structure and sarcomeric layout of the Z-disc and I-band region of human elastic (soleus) titin and predicted alignment with the Drosophila TITIN protein. Immunoglobulin-like domains (blue), interdomain sequences (red), and the FN3 domains (white) are shown. The single elastic PEVK domain (yellow) of the human titin consists of 70% proline (P), glutamic acid (E), valine (V), and lysine (K). PEVK-1 (yellow) of D-TITIN (1,240 residues) consists of 58.5% P, E, V, and K; PEVK-2 (yellow) of D-TITIN (5,065 residues) consists of 52.4% P, E, V, and K.

Figure 1

Molecular characterization of the D-Titin gene. (A) Molecular map of the region in 62C containing D-Titin. A series of seven overlapping contigs span the entire D-Titin gene and link previously isolated D-Titin clones (KZ, NB, LG, JT) and four new D-Titin cDNAs (GH05716, CK340, CK55, LD28564). D-Titin spans from nucleotide (nt) 293742 (5′) to nt 182959 (3′) in Celera contig AE003473.1. The ORF in the LG clone was completely contained within the large contig, however; LG is a chimeric genomic clone that also includes noncoding sequences from cytological region 62B. The kettin sequence (Hakeda et al. 2000; Kolmerer et al. 2000) is entirely included within the D-Titin gene. Three P-element insertions in D-Titin, l(3)rL182, v(3)04860, and l(3)j1D7, are indicated, as well as the flanking DNA, isolated by plasmid rescue (PR4860 and PRj1D7). l(3)6265 is a P-element insertion used to clone flanking genomic DNA and map the proximal breakpoint of Df(3L)Aprt123. Df(3L)Aprt123 deletes the neighboring distal gene (mu2) but does not affect D-Titin function. (B) Protein expression profiles detected by an antibody against D-TITIN (α-KZ) (a–f) and an antibody against KET (Lakey et al. 1993) (g–l). Identical expression is detected in all the somatic (sm), visceral (vm), and pharyngeal muscles (ph) and their precursors during embryogenesis with both antibodies. (a and g) Stage 11 embryos, ventral view. (b and h) Stage 13 embryos, ventral view. (c and i) Stage 16 embryos, ventral view. (d and j) Stage 11 embryos, lateral view. (e and k) Stage 13 embryos, lateral view. (f and l) Stage 16 embryos, lateral view. All embryos are oriented with anterior to the left. For embryos shown in lateral view, dorsal is up. (C) D-Titin transcripts detected by whole-mount in situ hybridization. Genomic and cDNA clones mapping throughout the D-Titin gene (see gray lines in A) detected identical expression patterns throughout embryogenesis in all striated muscles and their precursors. Shown in a–d are embryos hybridized with the JT cDNA, which encodes a portion of the PEVK-2 domain. (a) ventral view of stage 13 embryo; (b) lateral view of stage 14 embryo; (c) ventral view of early stage 15 embryo; (d) lateral view of stage 16 embryo. (D) Domain structure and sarcomeric layout of the Z-disc and I-band region of human elastic (soleus) titin and predicted alignment with the Drosophila TITIN protein. Immunoglobulin-like domains (blue), interdomain sequences (red), and the FN3 domains (white) are shown. The single elastic PEVK domain (yellow) of the human titin consists of 70% proline (P), glutamic acid (E), valine (V), and lysine (K). PEVK-1 (yellow) of D-TITIN (1,240 residues) consists of 58.5% P, E, V, and K; PEVK-2 (yellow) of D-TITIN (5,065 residues) consists of 52.4% P, E, V, and K.

Figure 1

Molecular characterization of the D-Titin gene. (A) Molecular map of the region in 62C containing D-Titin. A series of seven overlapping contigs span the entire D-Titin gene and link previously isolated D-Titin clones (KZ, NB, LG, JT) and four new D-Titin cDNAs (GH05716, CK340, CK55, LD28564). D-Titin spans from nucleotide (nt) 293742 (5′) to nt 182959 (3′) in Celera contig AE003473.1. The ORF in the LG clone was completely contained within the large contig, however; LG is a chimeric genomic clone that also includes noncoding sequences from cytological region 62B. The kettin sequence (Hakeda et al. 2000; Kolmerer et al. 2000) is entirely included within the D-Titin gene. Three P-element insertions in D-Titin, l(3)rL182, v(3)04860, and l(3)j1D7, are indicated, as well as the flanking DNA, isolated by plasmid rescue (PR4860 and PRj1D7). l(3)6265 is a P-element insertion used to clone flanking genomic DNA and map the proximal breakpoint of Df(3L)Aprt123. Df(3L)Aprt123 deletes the neighboring distal gene (mu2) but does not affect D-Titin function. (B) Protein expression profiles detected by an antibody against D-TITIN (α-KZ) (a–f) and an antibody against KET (Lakey et al. 1993) (g–l). Identical expression is detected in all the somatic (sm), visceral (vm), and pharyngeal muscles (ph) and their precursors during embryogenesis with both antibodies. (a and g) Stage 11 embryos, ventral view. (b and h) Stage 13 embryos, ventral view. (c and i) Stage 16 embryos, ventral view. (d and j) Stage 11 embryos, lateral view. (e and k) Stage 13 embryos, lateral view. (f and l) Stage 16 embryos, lateral view. All embryos are oriented with anterior to the left. For embryos shown in lateral view, dorsal is up. (C) D-Titin transcripts detected by whole-mount in situ hybridization. Genomic and cDNA clones mapping throughout the D-Titin gene (see gray lines in A) detected identical expression patterns throughout embryogenesis in all striated muscles and their precursors. Shown in a–d are embryos hybridized with the JT cDNA, which encodes a portion of the PEVK-2 domain. (a) ventral view of stage 13 embryo; (b) lateral view of stage 14 embryo; (c) ventral view of early stage 15 embryo; (d) lateral view of stage 16 embryo. (D) Domain structure and sarcomeric layout of the Z-disc and I-band region of human elastic (soleus) titin and predicted alignment with the Drosophila TITIN protein. Immunoglobulin-like domains (blue), interdomain sequences (red), and the FN3 domains (white) are shown. The single elastic PEVK domain (yellow) of the human titin consists of 70% proline (P), glutamic acid (E), valine (V), and lysine (K). PEVK-1 (yellow) of D-TITIN (1,240 residues) consists of 58.5% P, E, V, and K; PEVK-2 (yellow) of D-TITIN (5,065 residues) consists of 52.4% P, E, V, and K.

Figure 1

Molecular characterization of the D-Titin gene. (A) Molecular map of the region in 62C containing D-Titin. A series of seven overlapping contigs span the entire D-Titin gene and link previously isolated D-Titin clones (KZ, NB, LG, JT) and four new D-Titin cDNAs (GH05716, CK340, CK55, LD28564). D-Titin spans from nucleotide (nt) 293742 (5′) to nt 182959 (3′) in Celera contig AE003473.1. The ORF in the LG clone was completely contained within the large contig, however; LG is a chimeric genomic clone that also includes noncoding sequences from cytological region 62B. The kettin sequence (Hakeda et al. 2000; Kolmerer et al. 2000) is entirely included within the D-Titin gene. Three P-element insertions in D-Titin, l(3)rL182, v(3)04860, and l(3)j1D7, are indicated, as well as the flanking DNA, isolated by plasmid rescue (PR4860 and PRj1D7). l(3)6265 is a P-element insertion used to clone flanking genomic DNA and map the proximal breakpoint of Df(3L)Aprt123. Df(3L)Aprt123 deletes the neighboring distal gene (mu2) but does not affect D-Titin function. (B) Protein expression profiles detected by an antibody against D-TITIN (α-KZ) (a–f) and an antibody against KET (Lakey et al. 1993) (g–l). Identical expression is detected in all the somatic (sm), visceral (vm), and pharyngeal muscles (ph) and their precursors during embryogenesis with both antibodies. (a and g) Stage 11 embryos, ventral view. (b and h) Stage 13 embryos, ventral view. (c and i) Stage 16 embryos, ventral view. (d and j) Stage 11 embryos, lateral view. (e and k) Stage 13 embryos, lateral view. (f and l) Stage 16 embryos, lateral view. All embryos are oriented with anterior to the left. For embryos shown in lateral view, dorsal is up. (C) D-Titin transcripts detected by whole-mount in situ hybridization. Genomic and cDNA clones mapping throughout the D-Titin gene (see gray lines in A) detected identical expression patterns throughout embryogenesis in all striated muscles and their precursors. Shown in a–d are embryos hybridized with the JT cDNA, which encodes a portion of the PEVK-2 domain. (a) ventral view of stage 13 embryo; (b) lateral view of stage 14 embryo; (c) ventral view of early stage 15 embryo; (d) lateral view of stage 16 embryo. (D) Domain structure and sarcomeric layout of the Z-disc and I-band region of human elastic (soleus) titin and predicted alignment with the Drosophila TITIN protein. Immunoglobulin-like domains (blue), interdomain sequences (red), and the FN3 domains (white) are shown. The single elastic PEVK domain (yellow) of the human titin consists of 70% proline (P), glutamic acid (E), valine (V), and lysine (K). PEVK-1 (yellow) of D-TITIN (1,240 residues) consists of 58.5% P, E, V, and K; PEVK-2 (yellow) of D-TITIN (5,065 residues) consists of 52.4% P, E, V, and K.

Figure 2

Localization of D-Titin to a cytogenetic interval containing only a single known gene (dre8). The upper left panel is a map of cytological region 62B-C showing the known genes that have been mapped to the region and the deficiencies used to map D-Titin. The remaining panels show in situ hybridization to polytene chromosomes with biotinylated D-Titin genomic phage clone 5 (Machado et al. 1998). The polytene chromosomes are from larvae heterozygous for each deficiency. D-Titin is deleted in Df(3L)Aprt143 and Df(3L)Aprt24. D-Titin is not deleted in Df(3L)Aprt18, Df(3L)Aprt123, and Df(3L)Aprt126. Identical hybridization results were obtained using D-Titin clones that mapped throughout the D-Titin gene. (d, distal; p, proximal).

Figure 4

Mitotic phenotypes in D-Titin mutants. Mitotic figures of wild-type and D-Titin mutants from untreated brain squashes of third instar larvae. Brains were stained with Hoechst. (A) Low magnification view of wild-type neuroblasts showing a field of uniformly-sized cells in interphase. (B) Higher magnification of wild-type neuroblasts showing a field of interphase cells. Arrow indicates a cell in anaphase. (C) Low magnification view of titin neuroblasts. Note large polyploid nuclei (white star) and large polyploid nuclei with variably condensed chromatin and chromosome breaks (small white arrows). (D) Higher magnification of a titin neuroblast showing variably condensed chromatin in addition to chromosome breakage. (E and F) Low and high magnification views of titin neuroblasts with polyploid nuclei with highly condensed chromatin (stars) and polyploid nuclei with variable chromosome condensation (arrows). Images were viewed and photographed under 100× objective, 10× eyepiece.

Figure 3

Muscle disorganization in D-Titin mutants. Myoblast fusion and gut defects in D-Titin mutants. Wild-type (a and d) and mutant (b, c, e, and f) embryos were stained with a D-TITIN antibody (α-KZ) (Machado et al. 1998). The mutations cause unfused myoblasts (b and c, arrows), disorganized muscle (b, c, e, and f), and gut morphogenesis defects (e and f, arrows). All embryos are oriented with anterior to the left. (a–c) are lateral views, dorsal is up. (d–f) are ventral views. (b and e) Titin⁸ homozygotes; (c) Titin⁵ homozygote; (f) Titin⁴ homozygote.

Figure 5

Chromosomal defects in D-Titin mutants. Brains were treated with colchicine/hypotonic shock and stained with orcein. (A) Wild-type neuroblast showing well spread, condensed, diploid chromosomes characteristic of those treated with colchicine and hypotonic shock. (B) Two neuroblasts from a titin ¹¹ homozygous larva showing one cell with severe chromosome undercondensation (black arrow) and an adjacent cell with relatively normal levels of chromosome condensation (white arrow). (C) A neuroblast from a titin ¹/titin ¹⁰ larva showing chromosome undercondensation. (D) Two neuroblasts from a titin ¹¹ homozygous larva showing one cell with undercondensation of the centromeric heterochromatin (black arrow) and another cell with relatively normal chromosomes. (E) A neuroblast from a titin ⁸ homozygous larva showing chromosome decondensation and premature sister chromatid separation. The black arrow indicates one of the large autosomes where the two sister chromosomes have completely separated. (F) A neuroblast from a titin homozygous larva showing premature sister chromatid separation and decondensation of the centromeric heterochromatin (black arrow). (G) A neuroblast from a titin homozygous larva showing polyploidy, premature sister chromatid separation (for example the chromosomes indicated with the large black arrow) and decondensation of centromeric heterochromatin (small black arrows). (H) A neuroblast from a titin ¹/titin ⁸ larva showing normal levels of condensation but missing the two sex chrosomes. Images were viewed and photographed under 100× objective, 10× eyepiece.