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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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Depletion of ALMS1 causes centrosome splitting. (A) Cyclin B1 immunostaining was used to determine cell cycle stage, allowing cells entering mitosis to be excluded from analysis. Examples of control cells at the G2-M transition and in G1/early S phase are shown. Centrioles were visualized by costaining with an antibody to acetylated tubulin. Note the separating centrosomes (each containing one parental and one progeny centriole) at G2-M and the close association of the two parental centrioles in G1/early S phase. (B) Immunofluorescence microscopy analysis of centrosome splitting in G1/early S phase cells. Cells were treated with the indicated siRNAs and costained with antibodies to cyclin B1 and acetylated tubulin. Interphase cells with undetectable cyclin B1 and with centrioles >2 μm apart were classed as having a split centrosome. (C) Quantification of centrosome splitting, after siRNA treatment, in interphase cells with undetectable cyclin B1. Depletion of the centrosome cohesion protein C-Nap1 was used as a positive control. The mean ± SE of three independent experiments is shown; in each experiment at least 100 cells were counted for each condition. (D) Model of ALMS1 function in centrosome cohesion, based on the model of C-Nap1/rootletin–dependent centrosome cohesion proposed by Bahe et al. (2005). Depletion of ALMS1 causes, by an unknown mechanism, reductions in the level of C-Nap1 at centrioles. This is predicted to perturb docking of interconnecting or “entangling” fibers with the proximal end of each centriole, leading to centrosome splitting. Procentrioles, which assemble orthogonally to parental centrioles during S and G2 phase, are depicted by dashed lines. Note that C-Nap1 is not thought to localize to the interface between the procentriole and centriole or be involved in maintaining their attachment (Mayor et al., 2000); it is not known if ALMS1 localizes to this interface.
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Figure 9: Depletion of ALMS1 causes centrosome splitting. (A) Cyclin B1 immunostaining was used to determine cell cycle stage, allowing cells entering mitosis to be excluded from analysis. Examples of control cells at the G2-M transition and in G1/early S phase are shown. Centrioles were visualized by costaining with an antibody to acetylated tubulin. Note the separating centrosomes (each containing one parental and one progeny centriole) at G2-M and the close association of the two parental centrioles in G1/early S phase. (B) Immunofluorescence microscopy analysis of centrosome splitting in G1/early S phase cells. Cells were treated with the indicated siRNAs and costained with antibodies to cyclin B1 and acetylated tubulin. Interphase cells with undetectable cyclin B1 and with centrioles >2 μm apart were classed as having a split centrosome. (C) Quantification of centrosome splitting, after siRNA treatment, in interphase cells with undetectable cyclin B1. Depletion of the centrosome cohesion protein C-Nap1 was used as a positive control. The mean ± SE of three independent experiments is shown; in each experiment at least 100 cells were counted for each condition. (D) Model of ALMS1 function in centrosome cohesion, based on the model of C-Nap1/rootletin–dependent centrosome cohesion proposed by Bahe et al. (2005). Depletion of ALMS1 causes, by an unknown mechanism, reductions in the level of C-Nap1 at centrioles. This is predicted to perturb docking of interconnecting or “entangling” fibers with the proximal end of each centriole, leading to centrosome splitting. Procentrioles, which assemble orthogonally to parental centrioles during S and G2 phase, are depicted by dashed lines. Note that C-Nap1 is not thought to localize to the interface between the procentriole and centriole or be involved in maintaining their attachment (Mayor et al., 2000); it is not known if ALMS1 localizes to this interface.

Mentions: Next we examined the effect of ALMS1 depletion on cohesion between parental centrioles, using direct depletion of C-Nap1 as a positive control. We chose to analyze cells in G1 to avoid including cells undergoing the physiological process of centrosome separation at the G2-M transition and also because siRNA-mediated depletion of C-Nap1 has been reported to induce G1-S arrest (Mikule et al., 2007). We used cyclin B1 as a marker for cell cycle stage (Figure 9A). Cyclin B1 is undetectable in G1, begins to accumulate in the cytoplasm in S phase, and then at the centrosome in late S/early G2 phase, redistributes to the nucleus in prophase and is degraded at the metaphase–anaphase transition (Pines and Hunter, 1991; Bailly et al., 1992). In certain cell lines the immature parental centriole has been reported to be relatively free to move within the cytoplasm in G1 (Piel et al., 2000); however, we found that in ∼95% of interphase cyclin B1–negative hTERT-RPE1 cells the two centrioles were closely associated (Figure 9, A–C). Furthermore, treatment with C-Nap1–directed siRNA led to a dramatic increase in the distance between centrioles in ∼65% of these cells, indicating that these conditions were suitable for analyzing C-Nap1–dependent association of centrioles (Figure 9, B and C). Consistent with reduced levels of C-Nap1 at the centrioles of ALMS1-depleted cells, we noted an increase in the proportion of these cells with centrioles separated by >2 μm (referred to here as split centrosomes; Figure 9, B and C). The increase was modest compared with that observed in cells treated with C-Nap1–directed siRNA, possibly because of higher residual levels of C-Nap1 at the centrosome in ALMS1-depleted cells (Supplemental Figure S7C).


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)

Depletion of ALMS1 causes centrosome splitting. (A) Cyclin B1 immunostaining was used to determine cell cycle stage, allowing cells entering mitosis to be excluded from analysis. Examples of control cells at the G2-M transition and in G1/early S phase are shown. Centrioles were visualized by costaining with an antibody to acetylated tubulin. Note the separating centrosomes (each containing one parental and one progeny centriole) at G2-M and the close association of the two parental centrioles in G1/early S phase. (B) Immunofluorescence microscopy analysis of centrosome splitting in G1/early S phase cells. Cells were treated with the indicated siRNAs and costained with antibodies to cyclin B1 and acetylated tubulin. Interphase cells with undetectable cyclin B1 and with centrioles >2 μm apart were classed as having a split centrosome. (C) Quantification of centrosome splitting, after siRNA treatment, in interphase cells with undetectable cyclin B1. Depletion of the centrosome cohesion protein C-Nap1 was used as a positive control. The mean ± SE of three independent experiments is shown; in each experiment at least 100 cells were counted for each condition. (D) Model of ALMS1 function in centrosome cohesion, based on the model of C-Nap1/rootletin–dependent centrosome cohesion proposed by Bahe et al. (2005). Depletion of ALMS1 causes, by an unknown mechanism, reductions in the level of C-Nap1 at centrioles. This is predicted to perturb docking of interconnecting or “entangling” fibers with the proximal end of each centriole, leading to centrosome splitting. Procentrioles, which assemble orthogonally to parental centrioles during S and G2 phase, are depicted by dashed lines. Note that C-Nap1 is not thought to localize to the interface between the procentriole and centriole or be involved in maintaining their attachment (Mayor et al., 2000); it is not known if ALMS1 localizes to this interface.
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Figure 9: Depletion of ALMS1 causes centrosome splitting. (A) Cyclin B1 immunostaining was used to determine cell cycle stage, allowing cells entering mitosis to be excluded from analysis. Examples of control cells at the G2-M transition and in G1/early S phase are shown. Centrioles were visualized by costaining with an antibody to acetylated tubulin. Note the separating centrosomes (each containing one parental and one progeny centriole) at G2-M and the close association of the two parental centrioles in G1/early S phase. (B) Immunofluorescence microscopy analysis of centrosome splitting in G1/early S phase cells. Cells were treated with the indicated siRNAs and costained with antibodies to cyclin B1 and acetylated tubulin. Interphase cells with undetectable cyclin B1 and with centrioles >2 μm apart were classed as having a split centrosome. (C) Quantification of centrosome splitting, after siRNA treatment, in interphase cells with undetectable cyclin B1. Depletion of the centrosome cohesion protein C-Nap1 was used as a positive control. The mean ± SE of three independent experiments is shown; in each experiment at least 100 cells were counted for each condition. (D) Model of ALMS1 function in centrosome cohesion, based on the model of C-Nap1/rootletin–dependent centrosome cohesion proposed by Bahe et al. (2005). Depletion of ALMS1 causes, by an unknown mechanism, reductions in the level of C-Nap1 at centrioles. This is predicted to perturb docking of interconnecting or “entangling” fibers with the proximal end of each centriole, leading to centrosome splitting. Procentrioles, which assemble orthogonally to parental centrioles during S and G2 phase, are depicted by dashed lines. Note that C-Nap1 is not thought to localize to the interface between the procentriole and centriole or be involved in maintaining their attachment (Mayor et al., 2000); it is not known if ALMS1 localizes to this interface.
Mentions: Next we examined the effect of ALMS1 depletion on cohesion between parental centrioles, using direct depletion of C-Nap1 as a positive control. We chose to analyze cells in G1 to avoid including cells undergoing the physiological process of centrosome separation at the G2-M transition and also because siRNA-mediated depletion of C-Nap1 has been reported to induce G1-S arrest (Mikule et al., 2007). We used cyclin B1 as a marker for cell cycle stage (Figure 9A). Cyclin B1 is undetectable in G1, begins to accumulate in the cytoplasm in S phase, and then at the centrosome in late S/early G2 phase, redistributes to the nucleus in prophase and is degraded at the metaphase–anaphase transition (Pines and Hunter, 1991; Bailly et al., 1992). In certain cell lines the immature parental centriole has been reported to be relatively free to move within the cytoplasm in G1 (Piel et al., 2000); however, we found that in ∼95% of interphase cyclin B1–negative hTERT-RPE1 cells the two centrioles were closely associated (Figure 9, A–C). Furthermore, treatment with C-Nap1–directed siRNA led to a dramatic increase in the distance between centrioles in ∼65% of these cells, indicating that these conditions were suitable for analyzing C-Nap1–dependent association of centrioles (Figure 9, B and C). Consistent with reduced levels of C-Nap1 at the centrioles of ALMS1-depleted cells, we noted an increase in the proportion of these cells with centrioles separated by >2 μm (referred to here as split centrosomes; Figure 9, B and C). The increase was modest compared with that observed in cells treated with C-Nap1–directed siRNA, possibly because of higher residual levels of C-Nap1 at the centrosome in ALMS1-depleted cells (Supplemental Figure S7C).

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