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A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends

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ABSTRACT

The Astrin/SKAP complex regulates mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, Kern et al. demonstrate that a previously unappreciated short SKAP isoform mediates mitotic spindle positioning at astral microtubule plus ends.

No MeSH data available.


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SKAP has distinct mitotic and testis-specific isoforms. (A) Western blot showing the molecular mass of endogenous SKAP from HeLa cells assessed by protein marker migration. (B, top) Immunoprecipitation mass spectrometry (IP-MS) data pooled from LAP-Astrin IPs analyzed for Astrin and SKAP peptides. (bottom) Identified SKAP peptides mapped against the SKAP amino acid sequence (ID: Q9Y448-1). (C, left) Map of the 5′ end of the SKAP locus with RNA-sequencing reads from Human BodyMap 2.0. (right) Schematic of the transcript for the short (mitotic) SKAP isoform. The underlined lysine indicates the first peptide identifiable in a tryptic digest for short SKAP. (D) Confocal image of long (testis) SKAP IF localization to elongating spermatids in a mouse seminiferous (Stage I) tubule. The seminiferous tubule lumen is indicated. (top right) Zoom-in of elongating spermatids. (E) Testis section showing IF long (testis) SKAP localization to elongating spermatids in a mouse seminiferous tubule transitioning from developmental Stage X to XI. (left) Tubule region imaged with a 40× objective. (right) Boxed region (Stage X) imaged using a 100× objective to indicate DNA localized puncta (∼6× zoom). Bars, 20 µm.
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fig1: SKAP has distinct mitotic and testis-specific isoforms. (A) Western blot showing the molecular mass of endogenous SKAP from HeLa cells assessed by protein marker migration. (B, top) Immunoprecipitation mass spectrometry (IP-MS) data pooled from LAP-Astrin IPs analyzed for Astrin and SKAP peptides. (bottom) Identified SKAP peptides mapped against the SKAP amino acid sequence (ID: Q9Y448-1). (C, left) Map of the 5′ end of the SKAP locus with RNA-sequencing reads from Human BodyMap 2.0. (right) Schematic of the transcript for the short (mitotic) SKAP isoform. The underlined lysine indicates the first peptide identifiable in a tryptic digest for short SKAP. (D) Confocal image of long (testis) SKAP IF localization to elongating spermatids in a mouse seminiferous (Stage I) tubule. The seminiferous tubule lumen is indicated. (top right) Zoom-in of elongating spermatids. (E) Testis section showing IF long (testis) SKAP localization to elongating spermatids in a mouse seminiferous tubule transitioning from developmental Stage X to XI. (left) Tubule region imaged with a 40× objective. (right) Boxed region (Stage X) imaged using a 100× objective to indicate DNA localized puncta (∼6× zoom). Bars, 20 µm.

Mentions: All previous studies on SKAP/KNSTRN have used a consensus annotated database protein sequence (ID: Q9Y448-1) with a predicted molecular mass of 34.5 kD. However, in analyzing the behavior of SKAP in human tissue culture cells, our affinity-purified anti-SKAP antibody detected a protein of ∼27 kD by SDS-PAGE and Western blotting (Fig. 1 A). Based on mass spectrometry analysis of endogenous SKAP isolated from HeLa cells, we were unable to detect peptides from a large region of the N terminus for the annotated SKAP protein (Fig. 1 B). In addition, in RNA-sequencing data from the Human BodyMap 2.0 database, we found that the only tissue with reads spanning the entire annotated SKAP sequence was testis (Fig. 1 C). Indeed, although we were unable to identify peptides corresponding to the annotated SKAP N terminus in mitotic cells based on a mass spectrometry analysis, immunoprecipitation (IP) of SKAP from adult mouse testes identified peptides corresponding to this N-terminal region, as well as copurifying peptides from Astrin (Fig. S1 A). In all other tissues, transcriptional initiation began within the first annotated exon, resulting in an mRNA lacking the previously defined start codon (Fig. 1 C; also see E-MTAB-513 in ArrayExpress: http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513). Instead, the first in-frame, coding methionine appeared within the previously defined exon 2. The shorter SKAP isoform generated using this downstream start codon has a predicted molecular mass of 26.9 kD, consistent with our mass spectrometry and Western blot analysis (Fig. 1, A and B; and Fig. S1 B). Although SKAP and its binding partner Astrin are conserved throughout vertebrates (Fig. S1 C), the longer, testis-specific SKAP isoform is present only in eutherian mammals (Fig. S1, B and C).


A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends
SKAP has distinct mitotic and testis-specific isoforms. (A) Western blot showing the molecular mass of endogenous SKAP from HeLa cells assessed by protein marker migration. (B, top) Immunoprecipitation mass spectrometry (IP-MS) data pooled from LAP-Astrin IPs analyzed for Astrin and SKAP peptides. (bottom) Identified SKAP peptides mapped against the SKAP amino acid sequence (ID: Q9Y448-1). (C, left) Map of the 5′ end of the SKAP locus with RNA-sequencing reads from Human BodyMap 2.0. (right) Schematic of the transcript for the short (mitotic) SKAP isoform. The underlined lysine indicates the first peptide identifiable in a tryptic digest for short SKAP. (D) Confocal image of long (testis) SKAP IF localization to elongating spermatids in a mouse seminiferous (Stage I) tubule. The seminiferous tubule lumen is indicated. (top right) Zoom-in of elongating spermatids. (E) Testis section showing IF long (testis) SKAP localization to elongating spermatids in a mouse seminiferous tubule transitioning from developmental Stage X to XI. (left) Tubule region imaged with a 40× objective. (right) Boxed region (Stage X) imaged using a 100× objective to indicate DNA localized puncta (∼6× zoom). Bars, 20 µm.
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Related In: Results  -  Collection

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fig1: SKAP has distinct mitotic and testis-specific isoforms. (A) Western blot showing the molecular mass of endogenous SKAP from HeLa cells assessed by protein marker migration. (B, top) Immunoprecipitation mass spectrometry (IP-MS) data pooled from LAP-Astrin IPs analyzed for Astrin and SKAP peptides. (bottom) Identified SKAP peptides mapped against the SKAP amino acid sequence (ID: Q9Y448-1). (C, left) Map of the 5′ end of the SKAP locus with RNA-sequencing reads from Human BodyMap 2.0. (right) Schematic of the transcript for the short (mitotic) SKAP isoform. The underlined lysine indicates the first peptide identifiable in a tryptic digest for short SKAP. (D) Confocal image of long (testis) SKAP IF localization to elongating spermatids in a mouse seminiferous (Stage I) tubule. The seminiferous tubule lumen is indicated. (top right) Zoom-in of elongating spermatids. (E) Testis section showing IF long (testis) SKAP localization to elongating spermatids in a mouse seminiferous tubule transitioning from developmental Stage X to XI. (left) Tubule region imaged with a 40× objective. (right) Boxed region (Stage X) imaged using a 100× objective to indicate DNA localized puncta (∼6× zoom). Bars, 20 µm.
Mentions: All previous studies on SKAP/KNSTRN have used a consensus annotated database protein sequence (ID: Q9Y448-1) with a predicted molecular mass of 34.5 kD. However, in analyzing the behavior of SKAP in human tissue culture cells, our affinity-purified anti-SKAP antibody detected a protein of ∼27 kD by SDS-PAGE and Western blotting (Fig. 1 A). Based on mass spectrometry analysis of endogenous SKAP isolated from HeLa cells, we were unable to detect peptides from a large region of the N terminus for the annotated SKAP protein (Fig. 1 B). In addition, in RNA-sequencing data from the Human BodyMap 2.0 database, we found that the only tissue with reads spanning the entire annotated SKAP sequence was testis (Fig. 1 C). Indeed, although we were unable to identify peptides corresponding to the annotated SKAP N terminus in mitotic cells based on a mass spectrometry analysis, immunoprecipitation (IP) of SKAP from adult mouse testes identified peptides corresponding to this N-terminal region, as well as copurifying peptides from Astrin (Fig. S1 A). In all other tissues, transcriptional initiation began within the first annotated exon, resulting in an mRNA lacking the previously defined start codon (Fig. 1 C; also see E-MTAB-513 in ArrayExpress: http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513). Instead, the first in-frame, coding methionine appeared within the previously defined exon 2. The shorter SKAP isoform generated using this downstream start codon has a predicted molecular mass of 26.9 kD, consistent with our mass spectrometry and Western blot analysis (Fig. 1, A and B; and Fig. S1 B). Although SKAP and its binding partner Astrin are conserved throughout vertebrates (Fig. S1 C), the longer, testis-specific SKAP isoform is present only in eutherian mammals (Fig. S1, B and C).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

The Astrin/SKAP complex regulates mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, Kern et al. demonstrate that a previously unappreciated short SKAP isoform mediates mitotic spindle positioning at astral microtubule plus ends.

No MeSH data available.


Related in: MedlinePlus