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Centriolar association of ALMS1 and likely centrosomal functions of the ALMS motif-containing proteins C10orf90 and KIAA1731.

Knorz VJ, Spalluto C, Lessard M, Purvis TL, Adigun FF, Collin GB, Hanley NA, Wilson DI, Hearn T - Mol. Biol. Cell (2010)

Bottom Line: We also show that ALMS1 localizes specifically to the proximal ends of centrioles and basal bodies, where it colocalizes with the centrosome cohesion protein C-Nap1.RNAi analysis reveals markedly diminished centrosomal levels of C-Nap1 and compromised cohesion of parental centrioles in ALMS1-depleted cells.In summary, these data suggest centrosomal functions for C10orf90 and KIAA1731 and new centriole-related functions for ALMS1.

View Article: PubMed Central - PubMed

Affiliation: Centre for Human Development, Stem Cells and Regeneration, Human Genetics Division, University of Southampton, Southampton, United Kingdom.

ABSTRACT
Mutations in the human gene ALMS1 cause Alström syndrome, a rare progressive condition characterized by neurosensory degeneration and metabolic defects. ALMS1 protein localizes to the centrosome and has been implicated in the assembly and/or maintenance of primary cilia; however its precise function, distribution within the centrosome, and mechanism of centrosomal recruitment are unknown. The C-terminus of ALMS1 contains a region with similarity to the uncharacterized human protein C10orf90, termed the ALMS motif. Here, we show that a third human protein, the candidate centrosomal protein KIAA1731, contains an ALMS motif and that exogenously expressed KIAA1731 and C10orf90 localize to the centrosome. However, based on deletion analysis of ALMS1, the ALMS motif appears unlikely to be critical for centrosomal targeting. RNAi analyses suggest that C10orf90 and KIAA1731 have roles in primary cilium assembly and centriole formation/stability, respectively. We also show that ALMS1 localizes specifically to the proximal ends of centrioles and basal bodies, where it colocalizes with the centrosome cohesion protein C-Nap1. RNAi analysis reveals markedly diminished centrosomal levels of C-Nap1 and compromised cohesion of parental centrioles in ALMS1-depleted cells. In summary, these data suggest centrosomal functions for C10orf90 and KIAA1731 and new centriole-related functions for ALMS1.

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siRNA-mediated depletion of KIAA1731 leads to loss of centrosome markers in hTERT-RPE1 cells. (A) qRT-PCR analysis showing depletion of KIAA1731 mRNA by two different siRNA duplexes. Results are expressed relative to the negative control siRNA and represent the mean ± SD of triplicate assays. (B) Quantification of cells in which immunostaining of centriolar acetylated tubulin and either ALMS1 or γ-tubulin was undetectable. The mean of three independent experiments is shown; in each experiment 100–300 cells were counted for each condition. Error bars, SE. (C) Examples of siRNA-treated cells costained with antibodies to ALMS1 and acetylated tubulin. The middle and bottom panels show cells with markedly diminished and undetectable immunostaining, respectively. (D) siRNA-treated cells costained with antibodies against C-Nap1 and pericentrin, showing loss of both markers in a cell treated with KIAA1731-directed siRNA. Size bars, 5 μm.
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Figure 4: siRNA-mediated depletion of KIAA1731 leads to loss of centrosome markers in hTERT-RPE1 cells. (A) qRT-PCR analysis showing depletion of KIAA1731 mRNA by two different siRNA duplexes. Results are expressed relative to the negative control siRNA and represent the mean ± SD of triplicate assays. (B) Quantification of cells in which immunostaining of centriolar acetylated tubulin and either ALMS1 or γ-tubulin was undetectable. The mean of three independent experiments is shown; in each experiment 100–300 cells were counted for each condition. Error bars, SE. (C) Examples of siRNA-treated cells costained with antibodies to ALMS1 and acetylated tubulin. The middle and bottom panels show cells with markedly diminished and undetectable immunostaining, respectively. (D) siRNA-treated cells costained with antibodies against C-Nap1 and pericentrin, showing loss of both markers in a cell treated with KIAA1731-directed siRNA. Size bars, 5 μm.

Mentions: Next we investigated the functions of KIAA1731 and C10orf90 by RNAi, targeting each with two different siRNA duplexes. In the absence of antibodies to the encoded proteins, siRNA-mediated knockdown was monitored at the mRNA level (Figures 4A and 6A). RT-PCR analysis confirmed expression of each gene but suggested that C10orf90 is expressed at a significantly lower level than KIAA1731 and ALMS1 in hTERT-RPE1 cells (our unpublished observation). Strikingly, a proportion of hTERT-RPE1 cells treated with KIAA1731-directed siRNAs displayed markedly reduced or undetectable centrosomal immunostaining for acetylated tubulin, ALMS1, C-Nap1, pericentrin, and γ-tubulin (Figure 4, B–D, and data not shown). Similar effects were observed in U2OS cells (data not shown). Loss of centriole (acetylated tubulin), centriole-associated (C-Nap1 and ALMS1; see below), and PCM (γ-tubulin and pericentrin) markers strongly suggests the absence of centrosomes. Because loss of centrioles can cause dispersal of the PCM (Bobinnec et al., 1998) and presumably also of centriole-associated proteins, these observations may be explained by a defect in centriole formation or stability.


Centriolar association of ALMS1 and likely centrosomal functions of the ALMS motif-containing proteins C10orf90 and KIAA1731.

Knorz VJ, Spalluto C, Lessard M, Purvis TL, Adigun FF, Collin GB, Hanley NA, Wilson DI, Hearn T - Mol. Biol. Cell (2010)

siRNA-mediated depletion of KIAA1731 leads to loss of centrosome markers in hTERT-RPE1 cells. (A) qRT-PCR analysis showing depletion of KIAA1731 mRNA by two different siRNA duplexes. Results are expressed relative to the negative control siRNA and represent the mean ± SD of triplicate assays. (B) Quantification of cells in which immunostaining of centriolar acetylated tubulin and either ALMS1 or γ-tubulin was undetectable. The mean of three independent experiments is shown; in each experiment 100–300 cells were counted for each condition. Error bars, SE. (C) Examples of siRNA-treated cells costained with antibodies to ALMS1 and acetylated tubulin. The middle and bottom panels show cells with markedly diminished and undetectable immunostaining, respectively. (D) siRNA-treated cells costained with antibodies against C-Nap1 and pericentrin, showing loss of both markers in a cell treated with KIAA1731-directed siRNA. Size bars, 5 μm.
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Figure 4: siRNA-mediated depletion of KIAA1731 leads to loss of centrosome markers in hTERT-RPE1 cells. (A) qRT-PCR analysis showing depletion of KIAA1731 mRNA by two different siRNA duplexes. Results are expressed relative to the negative control siRNA and represent the mean ± SD of triplicate assays. (B) Quantification of cells in which immunostaining of centriolar acetylated tubulin and either ALMS1 or γ-tubulin was undetectable. The mean of three independent experiments is shown; in each experiment 100–300 cells were counted for each condition. Error bars, SE. (C) Examples of siRNA-treated cells costained with antibodies to ALMS1 and acetylated tubulin. The middle and bottom panels show cells with markedly diminished and undetectable immunostaining, respectively. (D) siRNA-treated cells costained with antibodies against C-Nap1 and pericentrin, showing loss of both markers in a cell treated with KIAA1731-directed siRNA. Size bars, 5 μm.
Mentions: Next we investigated the functions of KIAA1731 and C10orf90 by RNAi, targeting each with two different siRNA duplexes. In the absence of antibodies to the encoded proteins, siRNA-mediated knockdown was monitored at the mRNA level (Figures 4A and 6A). RT-PCR analysis confirmed expression of each gene but suggested that C10orf90 is expressed at a significantly lower level than KIAA1731 and ALMS1 in hTERT-RPE1 cells (our unpublished observation). Strikingly, a proportion of hTERT-RPE1 cells treated with KIAA1731-directed siRNAs displayed markedly reduced or undetectable centrosomal immunostaining for acetylated tubulin, ALMS1, C-Nap1, pericentrin, and γ-tubulin (Figure 4, B–D, and data not shown). Similar effects were observed in U2OS cells (data not shown). Loss of centriole (acetylated tubulin), centriole-associated (C-Nap1 and ALMS1; see below), and PCM (γ-tubulin and pericentrin) markers strongly suggests the absence of centrosomes. Because loss of centrioles can cause dispersal of the PCM (Bobinnec et al., 1998) and presumably also of centriole-associated proteins, these observations may be explained by a defect in centriole formation or stability.

Bottom Line: We also show that ALMS1 localizes specifically to the proximal ends of centrioles and basal bodies, where it colocalizes with the centrosome cohesion protein C-Nap1.RNAi analysis reveals markedly diminished centrosomal levels of C-Nap1 and compromised cohesion of parental centrioles in ALMS1-depleted cells.In summary, these data suggest centrosomal functions for C10orf90 and KIAA1731 and new centriole-related functions for ALMS1.

View Article: PubMed Central - PubMed

Affiliation: Centre for Human Development, Stem Cells and Regeneration, Human Genetics Division, University of Southampton, Southampton, United Kingdom.

ABSTRACT
Mutations in the human gene ALMS1 cause Alström syndrome, a rare progressive condition characterized by neurosensory degeneration and metabolic defects. ALMS1 protein localizes to the centrosome and has been implicated in the assembly and/or maintenance of primary cilia; however its precise function, distribution within the centrosome, and mechanism of centrosomal recruitment are unknown. The C-terminus of ALMS1 contains a region with similarity to the uncharacterized human protein C10orf90, termed the ALMS motif. Here, we show that a third human protein, the candidate centrosomal protein KIAA1731, contains an ALMS motif and that exogenously expressed KIAA1731 and C10orf90 localize to the centrosome. However, based on deletion analysis of ALMS1, the ALMS motif appears unlikely to be critical for centrosomal targeting. RNAi analyses suggest that C10orf90 and KIAA1731 have roles in primary cilium assembly and centriole formation/stability, respectively. We also show that ALMS1 localizes specifically to the proximal ends of centrioles and basal bodies, where it colocalizes with the centrosome cohesion protein C-Nap1. RNAi analysis reveals markedly diminished centrosomal levels of C-Nap1 and compromised cohesion of parental centrioles in ALMS1-depleted cells. In summary, these data suggest centrosomal functions for C10orf90 and KIAA1731 and new centriole-related functions for ALMS1.

Show MeSH
Related in: MedlinePlus