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PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment

View Article: PubMed Central - PubMed

ABSTRACT

Zollo et al. report that mutations in PRUNE1, a phosphoesterase superfamily molecule, underlie primary microcephaly and profound global developmental delay in four unrelated families from Oman, India, Iran and Italy. The study highlights a potential role for prune during microtubule polymerization, suggesting that prune syndrome may be a tubulinopathy.

No MeSH data available.


Related in: MedlinePlus

PRUNE and tubulin. (A) Top: Cell-based microtubules co-sedimentation assay and SDS/PAGE analysis showing the binding of FLAG-tagged PRUNE to microtubule polymers (MT) using in vitro whole protein extracts from SHSY5Y clones overexpressing wild-type FLAG-tagged PRUNE or empty vector (as negative control), with immunoblotting with antibodies against anti-FLAG, anti-β-tubulin and anti-kinesin V (as a positive control due to its known binding to microtubule polymers). Wild-type FLAG-tagged PRUNE was found in the pellet (P) fraction in the presence of microtubule polymers, while it was found only in the supernatant (S) fraction in the absence of microtubule polymers, indicating microtubule binding. Bottom: Co-immunoprecipitation assay using Flag-tagged wild-type, D30N and R297W PRUNE protein expression in SHSY5Y inducible cell clones. The whole protein extract from empty vector (EV, as negative control), wild-type, D30N and R297W PRUNE-overexpressing cells incubated with antibodies against β-tubulin or α-tubulin to immunoprecipitate (IP), endogenous β-tubulin (left) or α-tubulin (right). A band of the expected size (60 kDa) was detected by western blotting using an anti-Flag antibody in the immunoprecipitate fraction from wild-type and D30N-overexpressing clones, indicating binding of PRUNE wild-type and D30N with both β- and α-tubulin. Flag-tagged R297W PRUNE was detected with a long exposure (Supplementary Fig. 2A and 2B). (B) Microtubule nucleation assay. SHSY5Y-inducible cells overexpressing wild-type, D30N, R297W PRUNE proteins were treated with doxycycline followed by immunofluorescence staining with β-tubulin antibody (red), and DAPI for DNA staining (blue). Cells containing microtubule asters with a diameter longer than 5 µm were scored and the results from a representative experiment in triplicate are shown. Left: The immunofluorescence analysis performed on the inducible clones after 2 min at 37°C showing some representative asters (in red) for each clone (Scale bars = 5 µm). The chart on the right indicates the percentage of cells with aster diameters longer than 5 µm. Wild-type PRUNE expressing clones show a higher percentage of cells containing asters longer than 5 µm, compared to those expressing D30N and R297W PRUNE (∼160 nuclei per clone were counted; Supplementary Table 3). (C) In vitro microtubule polymerization assay performed using wild-type (black), D30N (orange) and R297W (green) PRUNE purified from E. coli. The standard polymerization reaction, alone or in presence of the purified wild-type or mutated (D30N and R297W) PRUNE protein, incubated with tubulin and followed by absorbance readings at 360 nm (excitation at 360 nm, and emission at 420 nm; EnSpire manager software) to evaluate the maximum absolute curve slope. Polymerization curves are shown for the three phases of polymerization; I (nucleation), II (growth), III (steady state). The polymerization rate is enhanced (∼2-fold) in presence of wild-type PRUNE (black) in comparison with microtubules alone (blue). Polymerization in the presence of D30N PRUNE (orange) is unaffected, while it is unregulated by R297W PRUNE (green). Both mutations result in a notable delay of microtubule polymerization rate, which is particularly evident during the nucleation phase (phase I). The curves shown represent the average of n = 3 independent experiments, expressed as mean ± SD of samples assayed in triplicate. See Supplementary Fig. 3F for standard polymerization alone, and in the presence of 3 µM paclitaxel or 3 µM nocodazole, used as positive and negative controls, respectively.
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awx014-F3: PRUNE and tubulin. (A) Top: Cell-based microtubules co-sedimentation assay and SDS/PAGE analysis showing the binding of FLAG-tagged PRUNE to microtubule polymers (MT) using in vitro whole protein extracts from SHSY5Y clones overexpressing wild-type FLAG-tagged PRUNE or empty vector (as negative control), with immunoblotting with antibodies against anti-FLAG, anti-β-tubulin and anti-kinesin V (as a positive control due to its known binding to microtubule polymers). Wild-type FLAG-tagged PRUNE was found in the pellet (P) fraction in the presence of microtubule polymers, while it was found only in the supernatant (S) fraction in the absence of microtubule polymers, indicating microtubule binding. Bottom: Co-immunoprecipitation assay using Flag-tagged wild-type, D30N and R297W PRUNE protein expression in SHSY5Y inducible cell clones. The whole protein extract from empty vector (EV, as negative control), wild-type, D30N and R297W PRUNE-overexpressing cells incubated with antibodies against β-tubulin or α-tubulin to immunoprecipitate (IP), endogenous β-tubulin (left) or α-tubulin (right). A band of the expected size (60 kDa) was detected by western blotting using an anti-Flag antibody in the immunoprecipitate fraction from wild-type and D30N-overexpressing clones, indicating binding of PRUNE wild-type and D30N with both β- and α-tubulin. Flag-tagged R297W PRUNE was detected with a long exposure (Supplementary Fig. 2A and 2B). (B) Microtubule nucleation assay. SHSY5Y-inducible cells overexpressing wild-type, D30N, R297W PRUNE proteins were treated with doxycycline followed by immunofluorescence staining with β-tubulin antibody (red), and DAPI for DNA staining (blue). Cells containing microtubule asters with a diameter longer than 5 µm were scored and the results from a representative experiment in triplicate are shown. Left: The immunofluorescence analysis performed on the inducible clones after 2 min at 37°C showing some representative asters (in red) for each clone (Scale bars = 5 µm). The chart on the right indicates the percentage of cells with aster diameters longer than 5 µm. Wild-type PRUNE expressing clones show a higher percentage of cells containing asters longer than 5 µm, compared to those expressing D30N and R297W PRUNE (∼160 nuclei per clone were counted; Supplementary Table 3). (C) In vitro microtubule polymerization assay performed using wild-type (black), D30N (orange) and R297W (green) PRUNE purified from E. coli. The standard polymerization reaction, alone or in presence of the purified wild-type or mutated (D30N and R297W) PRUNE protein, incubated with tubulin and followed by absorbance readings at 360 nm (excitation at 360 nm, and emission at 420 nm; EnSpire manager software) to evaluate the maximum absolute curve slope. Polymerization curves are shown for the three phases of polymerization; I (nucleation), II (growth), III (steady state). The polymerization rate is enhanced (∼2-fold) in presence of wild-type PRUNE (black) in comparison with microtubules alone (blue). Polymerization in the presence of D30N PRUNE (orange) is unaffected, while it is unregulated by R297W PRUNE (green). Both mutations result in a notable delay of microtubule polymerization rate, which is particularly evident during the nucleation phase (phase I). The curves shown represent the average of n = 3 independent experiments, expressed as mean ± SD of samples assayed in triplicate. See Supplementary Fig. 3F for standard polymerization alone, and in the presence of 3 µM paclitaxel or 3 µM nocodazole, used as positive and negative controls, respectively.

Mentions: To gain further insight into the molecular mechanism by which PRUNE may regulate neurogenesis, we performed a mass-spectrometry-based interaction screen of proteins co-immunoprecipitated by wild-type PRUNE. This identified a number of different proteins involved in cytoskeleton organization as likely binding partners (data not shown). Among these, a putative interaction between PRUNE and tubulin, in particular β-tubulin, was most notable given previous studies highlighting the critical role of tubulins and microtubule-associated proteins during brain development, which may be mutated in autosomal recessive primary microcephaly (MCPH) (Woods et al., 2005; Bahi-Buisson et al., 2014; Sun and Hevner, 2014). We performed a cell-based microtubule-binding proteins spin-down assay (Darshan et al., 2011) with genetically modified SHSY5Y neuroblastoma cells to result in inducible clones overexpressing PRUNE wild-type or mutants, under a tetracycline inducible promoter (see ‘Material and methods’ section and Supplementary Fig. 3C). Upon induction with doxycycline we saw interaction of PRUNE with microtubules (Fig. 3A), which was not observed in the empty vector clone (kinesin V, a microtubules-associated protein, used as control; Fig. 3A). Next, co-immunoprecitation assays were performed in which endogenous tubulins, in particular β-and α-tubulin, were readily able to immunoprecipitate Flag-tagged full-length PRUNEFLAG (Fig. 3A). This interaction was found to be conserved with both PRUNE incorporating p.D30N and p.R297W mutations (PRUNED30N-FLAG, PRUNER297W-FLAG), although PRUNER297W-FLAG displayed less efficient binding (Supplementary Fig. 3A and B). Endogenous wild-type PRUNE protein subcellular localization was further evaluated by immunofluorescence analyses in HeLa cells during cell division, demonstrating overlap between PRUNE and of β-tubulin on astral and interpolar microtubules (prometaphase, metaphases, anaphase, and cytokinesis; Supplementary Fig. 4), indicative of a potentially important role in cellular division processes. To further investigate this role, we next explored a role of PRUNE in microtubule polymerization assay using GTP as substrate. Polymerization assays demonstrated that wild-type PRUNE as expressed in E. coli significantly enhances microtubule polymerization in nucleation, growth and steady-state (phase I-II-III; Fig. 3C and Supplementary Fig. 3F), and revealed a delay in microtubule formation affecting mainly growth phase associated with mutant PRUNE p.D30N protein, while PRUNE p.R297W negatively influences the early growth rate of microtubule polymerization processes, revealing distinct functional outcomes of each PRUNE mutation (Fig. 3C). Further, in studies in SH-SY5Y inducible clones expressing either wild-type or mutant (p.Asp30Asn and p.Arg297Trp) protein, mutant PRUNE-expressing cells were found to contain shortened microtubules compared with wild-type PRUNE, with aster diameter size measuring below 5 µm (Fig. 3B and Supplementary Table 3).Figure 3


PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment
PRUNE and tubulin. (A) Top: Cell-based microtubules co-sedimentation assay and SDS/PAGE analysis showing the binding of FLAG-tagged PRUNE to microtubule polymers (MT) using in vitro whole protein extracts from SHSY5Y clones overexpressing wild-type FLAG-tagged PRUNE or empty vector (as negative control), with immunoblotting with antibodies against anti-FLAG, anti-β-tubulin and anti-kinesin V (as a positive control due to its known binding to microtubule polymers). Wild-type FLAG-tagged PRUNE was found in the pellet (P) fraction in the presence of microtubule polymers, while it was found only in the supernatant (S) fraction in the absence of microtubule polymers, indicating microtubule binding. Bottom: Co-immunoprecipitation assay using Flag-tagged wild-type, D30N and R297W PRUNE protein expression in SHSY5Y inducible cell clones. The whole protein extract from empty vector (EV, as negative control), wild-type, D30N and R297W PRUNE-overexpressing cells incubated with antibodies against β-tubulin or α-tubulin to immunoprecipitate (IP), endogenous β-tubulin (left) or α-tubulin (right). A band of the expected size (60 kDa) was detected by western blotting using an anti-Flag antibody in the immunoprecipitate fraction from wild-type and D30N-overexpressing clones, indicating binding of PRUNE wild-type and D30N with both β- and α-tubulin. Flag-tagged R297W PRUNE was detected with a long exposure (Supplementary Fig. 2A and 2B). (B) Microtubule nucleation assay. SHSY5Y-inducible cells overexpressing wild-type, D30N, R297W PRUNE proteins were treated with doxycycline followed by immunofluorescence staining with β-tubulin antibody (red), and DAPI for DNA staining (blue). Cells containing microtubule asters with a diameter longer than 5 µm were scored and the results from a representative experiment in triplicate are shown. Left: The immunofluorescence analysis performed on the inducible clones after 2 min at 37°C showing some representative asters (in red) for each clone (Scale bars = 5 µm). The chart on the right indicates the percentage of cells with aster diameters longer than 5 µm. Wild-type PRUNE expressing clones show a higher percentage of cells containing asters longer than 5 µm, compared to those expressing D30N and R297W PRUNE (∼160 nuclei per clone were counted; Supplementary Table 3). (C) In vitro microtubule polymerization assay performed using wild-type (black), D30N (orange) and R297W (green) PRUNE purified from E. coli. The standard polymerization reaction, alone or in presence of the purified wild-type or mutated (D30N and R297W) PRUNE protein, incubated with tubulin and followed by absorbance readings at 360 nm (excitation at 360 nm, and emission at 420 nm; EnSpire manager software) to evaluate the maximum absolute curve slope. Polymerization curves are shown for the three phases of polymerization; I (nucleation), II (growth), III (steady state). The polymerization rate is enhanced (∼2-fold) in presence of wild-type PRUNE (black) in comparison with microtubules alone (blue). Polymerization in the presence of D30N PRUNE (orange) is unaffected, while it is unregulated by R297W PRUNE (green). Both mutations result in a notable delay of microtubule polymerization rate, which is particularly evident during the nucleation phase (phase I). The curves shown represent the average of n = 3 independent experiments, expressed as mean ± SD of samples assayed in triplicate. See Supplementary Fig. 3F for standard polymerization alone, and in the presence of 3 µM paclitaxel or 3 µM nocodazole, used as positive and negative controls, respectively.
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awx014-F3: PRUNE and tubulin. (A) Top: Cell-based microtubules co-sedimentation assay and SDS/PAGE analysis showing the binding of FLAG-tagged PRUNE to microtubule polymers (MT) using in vitro whole protein extracts from SHSY5Y clones overexpressing wild-type FLAG-tagged PRUNE or empty vector (as negative control), with immunoblotting with antibodies against anti-FLAG, anti-β-tubulin and anti-kinesin V (as a positive control due to its known binding to microtubule polymers). Wild-type FLAG-tagged PRUNE was found in the pellet (P) fraction in the presence of microtubule polymers, while it was found only in the supernatant (S) fraction in the absence of microtubule polymers, indicating microtubule binding. Bottom: Co-immunoprecipitation assay using Flag-tagged wild-type, D30N and R297W PRUNE protein expression in SHSY5Y inducible cell clones. The whole protein extract from empty vector (EV, as negative control), wild-type, D30N and R297W PRUNE-overexpressing cells incubated with antibodies against β-tubulin or α-tubulin to immunoprecipitate (IP), endogenous β-tubulin (left) or α-tubulin (right). A band of the expected size (60 kDa) was detected by western blotting using an anti-Flag antibody in the immunoprecipitate fraction from wild-type and D30N-overexpressing clones, indicating binding of PRUNE wild-type and D30N with both β- and α-tubulin. Flag-tagged R297W PRUNE was detected with a long exposure (Supplementary Fig. 2A and 2B). (B) Microtubule nucleation assay. SHSY5Y-inducible cells overexpressing wild-type, D30N, R297W PRUNE proteins were treated with doxycycline followed by immunofluorescence staining with β-tubulin antibody (red), and DAPI for DNA staining (blue). Cells containing microtubule asters with a diameter longer than 5 µm were scored and the results from a representative experiment in triplicate are shown. Left: The immunofluorescence analysis performed on the inducible clones after 2 min at 37°C showing some representative asters (in red) for each clone (Scale bars = 5 µm). The chart on the right indicates the percentage of cells with aster diameters longer than 5 µm. Wild-type PRUNE expressing clones show a higher percentage of cells containing asters longer than 5 µm, compared to those expressing D30N and R297W PRUNE (∼160 nuclei per clone were counted; Supplementary Table 3). (C) In vitro microtubule polymerization assay performed using wild-type (black), D30N (orange) and R297W (green) PRUNE purified from E. coli. The standard polymerization reaction, alone or in presence of the purified wild-type or mutated (D30N and R297W) PRUNE protein, incubated with tubulin and followed by absorbance readings at 360 nm (excitation at 360 nm, and emission at 420 nm; EnSpire manager software) to evaluate the maximum absolute curve slope. Polymerization curves are shown for the three phases of polymerization; I (nucleation), II (growth), III (steady state). The polymerization rate is enhanced (∼2-fold) in presence of wild-type PRUNE (black) in comparison with microtubules alone (blue). Polymerization in the presence of D30N PRUNE (orange) is unaffected, while it is unregulated by R297W PRUNE (green). Both mutations result in a notable delay of microtubule polymerization rate, which is particularly evident during the nucleation phase (phase I). The curves shown represent the average of n = 3 independent experiments, expressed as mean ± SD of samples assayed in triplicate. See Supplementary Fig. 3F for standard polymerization alone, and in the presence of 3 µM paclitaxel or 3 µM nocodazole, used as positive and negative controls, respectively.
Mentions: To gain further insight into the molecular mechanism by which PRUNE may regulate neurogenesis, we performed a mass-spectrometry-based interaction screen of proteins co-immunoprecipitated by wild-type PRUNE. This identified a number of different proteins involved in cytoskeleton organization as likely binding partners (data not shown). Among these, a putative interaction between PRUNE and tubulin, in particular β-tubulin, was most notable given previous studies highlighting the critical role of tubulins and microtubule-associated proteins during brain development, which may be mutated in autosomal recessive primary microcephaly (MCPH) (Woods et al., 2005; Bahi-Buisson et al., 2014; Sun and Hevner, 2014). We performed a cell-based microtubule-binding proteins spin-down assay (Darshan et al., 2011) with genetically modified SHSY5Y neuroblastoma cells to result in inducible clones overexpressing PRUNE wild-type or mutants, under a tetracycline inducible promoter (see ‘Material and methods’ section and Supplementary Fig. 3C). Upon induction with doxycycline we saw interaction of PRUNE with microtubules (Fig. 3A), which was not observed in the empty vector clone (kinesin V, a microtubules-associated protein, used as control; Fig. 3A). Next, co-immunoprecitation assays were performed in which endogenous tubulins, in particular β-and α-tubulin, were readily able to immunoprecipitate Flag-tagged full-length PRUNEFLAG (Fig. 3A). This interaction was found to be conserved with both PRUNE incorporating p.D30N and p.R297W mutations (PRUNED30N-FLAG, PRUNER297W-FLAG), although PRUNER297W-FLAG displayed less efficient binding (Supplementary Fig. 3A and B). Endogenous wild-type PRUNE protein subcellular localization was further evaluated by immunofluorescence analyses in HeLa cells during cell division, demonstrating overlap between PRUNE and of β-tubulin on astral and interpolar microtubules (prometaphase, metaphases, anaphase, and cytokinesis; Supplementary Fig. 4), indicative of a potentially important role in cellular division processes. To further investigate this role, we next explored a role of PRUNE in microtubule polymerization assay using GTP as substrate. Polymerization assays demonstrated that wild-type PRUNE as expressed in E. coli significantly enhances microtubule polymerization in nucleation, growth and steady-state (phase I-II-III; Fig. 3C and Supplementary Fig. 3F), and revealed a delay in microtubule formation affecting mainly growth phase associated with mutant PRUNE p.D30N protein, while PRUNE p.R297W negatively influences the early growth rate of microtubule polymerization processes, revealing distinct functional outcomes of each PRUNE mutation (Fig. 3C). Further, in studies in SH-SY5Y inducible clones expressing either wild-type or mutant (p.Asp30Asn and p.Arg297Trp) protein, mutant PRUNE-expressing cells were found to contain shortened microtubules compared with wild-type PRUNE, with aster diameter size measuring below 5 µm (Fig. 3B and Supplementary Table 3).Figure 3

View Article: PubMed Central - PubMed

ABSTRACT

Zollo et al. report that mutations in PRUNE1, a phosphoesterase superfamily molecule, underlie primary microcephaly and profound global developmental delay in four unrelated families from Oman, India, Iran and Italy. The study highlights a potential role for prune during microtubule polymerization, suggesting that prune syndrome may be a tubulinopathy.

No MeSH data available.


Related in: MedlinePlus