Author Correction: CEP120 interacts with C2CD3 and Talpid3 and is required for centriole appendage assembly and ciliogenesis.

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CEP120 interacts with C2CD3 and Talpid3 and is required for centriole appendage assembly and ciliogenesis.

Centrosomal protein 120 (CEP120) was originally identified as a daughter centriole-enriched protein that participates in centriole elongation. Recent studies showed that CEP120 gene mutations cause complex ciliopathy phenotypes in humans, including Joubert syndrome and Jeune asphyxiating thoracic dystrophy, suggesting that CEP120 plays an additional role in ciliogenesis. To investigate the potential roles of CEP120 in centriole elongation and cilia formation, we knocked out the CEP120 gene in p53-deficient RPE1 cells using the CRISPR/Cas9 editing system, and performed various analyses. We herein report that loss of CEP120 produces short centrioles with no apparent distal and subdistal appendages. CEP120 knockout was also associated with defective centriole elongation, impaired recruitment of C2CD3 and Talpid3 to the distal ends of centrioles, and consequent defects in centriole appendage assembly and cilia formation. Interestingly, wild-type CEP120 interacts with C2CD3 and Talpid3, whereas a disease-associated CEP120 mutant (I975S) has a low affinity for C2CD3 binding and perturbs cilia assembly. Together, our findings reveal a novel role of CEP120 in ciliogenesis by showing that it interacts with C2CD3 and Talpid3 to assemble centriole appendages and by illuminating the molecular mechanism through which the CEP120 (I975S) mutation causes complex ciliopathies.

CEP295 interacts with microtubules and is required for centriole elongation.

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules.

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis.

CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity.

CEP120 interacts with CPAP and positively regulates centriole elongation.

Centriole duplication begins with the formation of a single procentriole next to a preexisting centriole. CPAP (centrosomal protein 4.1-associated protein) was previously reported to participate in centriole elongation. Here, we show that CEP120 is a cell cycle-regulated protein that directly interacts with CPAP and is required for centriole duplication. CEP120 levels increased gradually from early S to G2/M and decreased significantly after mitosis. Forced overexpression of either CEP120 or CPAP not only induced the assembly of overly long centrioles but also produced atypical supernumerary centrioles that grew from these long centrioles. Depletion of CEP120 inhibited CPAP-induced centriole elongation and vice versa, implying that these proteins work together to regulate centriole elongation. Furthermore, CEP120 was found to contain an N-terminal microtubule-binding domain, a C-terminal dimerization domain, and a centriolar localization domain. Overexpression of a microtubule binding-defective CEP120-K76A mutant significantly suppressed the formation of elongated centrioles. Together, our results indicate that CEP120 is a CPAP-interacting protein that positively regulates centriole elongation.

Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly.

Centrioles are cylindrical structures that are usually composed of nine triplets of microtubules (MTs) organized around a cartwheel-shaped structure. Recent studies have proposed a structural model of the SAS-6-based cartwheel, yet we do not know the molecular detail of how the cartwheel participates in centriolar MT assembly. In this study, we demonstrate that the human microcephaly protein, CEP135, directly interacts with hSAS-6 via its carboxyl-terminus and with MTs via its amino-terminus. Unexpectedly, CEP135 also interacts with another microcephaly protein CPAP via its amino terminal domain. Depletion of CEP135 not only perturbed the centriolar localization of CPAP, but also blocked CPAP-induced centriole elongation. Furthermore, CEP135 depletion led to abnormal centriole structures with altered numbers of MT triplets and shorter centrioles. Overexpression of a CEP135 mutant lacking the proper interaction with hSAS-6 had a dominant-negative effect on centriole assembly. We propose that CEP135 may serve as a linker protein that directly connects the central hub protein, hSAS-6, to the outer MTs, and suggest that this interaction stabilizes the proper cartwheel structure for further CPAP-mediated centriole elongation.

SV40 T/t-common polypeptide enhances the sensitivity of HER2-overexpressing human cancer cells to anticancer drugs cisplatin and doxorubicin.

HER2-overexpressing cancer cells are resistant to cisplatin (CDDP) and doxorubicin (DXR). Here we report that SV40 T/t-common polypeptide could specifically sensitize HER2-overexpressing cancer cells to CDDP and DXR and specifically enhance CDDP- or DXR-induced apoptosis in these cells. This activity of T/t-common may be attributed to its ability to inhibit Bcl-2 and Bcl-XL and to suppress ERK activity in CDDP- or DXR-treated HER2-overexpressing cancer cells. T/t-common could enhance the antitumor activity of DXR on HER2-overexpressing ovarian tumor in NOD/SCID mice, suggesting that combination therapy using T/t-common and chemotherapeutic agents may provide a new approach for treating HER2-overexpressing cancers.

The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.

Centriole duplication involves the growth of a procentriole next to the parental centriole. Mutations in STIL and CPAP/CENPJ cause primary microcephaly (MCPH). Here, we show that human STIL has an asymmetric localization to the daughter centriole and is required for procentriole formation. STIL levels oscillate during the cell cycle. Interestingly, STIL interacts directly with CPAP and forms a complex with hSAS6. A natural mutation of CPAP (E1235V) that causes MCPH in humans leads to significantly lower binding to STIL. Overexpression of STIL induced the formation of multiple procentrioles around the parental centriole. STIL depletion inhibited normal centriole duplication, Plk4-induced centriole amplification, and CPAP-induced centriole elongation, and resulted in a failure to localize hSAS6 and CPAP to the base of the nascent procentriole. Furthermore, hSAS6 depletion hindered STIL targeting to the procentriole, implying that STIL and hSAS6 are mutually dependent for their centriolar localization. Together, our results indicate that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH.

CPAP is a cell-cycle regulated protein that controls centriole length.

Centriole duplication involves the growing of a procentriole (progeny centriole) next to the proximal end of each pre-existing centriole (parental centriole). The molecular mechanisms that regulate procentriole elongation remain obscure. We show here that expression of the centriolar protein CPAP (centrosomal P4.1-associated protein) is carefully regulated during the cell cycle, with the protein being degraded in late mitosis. Depletion of CPAP inhibited centrosome duplication, whereas excess CPAP induced the formation of elongated procentriole-like structures (PLSs), which contain stable microtubules and several centriolar proteins. Ultrastructural analysis revealed that these structures are similar to procentrioles with elongated microtubules. Overexpression of a CPAP mutant (CPAP-377EE) that does not bind to tubulin dimers significantly inhibited the formation of CPAP-induced PLSs. Together, these results suggest that CPAP is a new regulator of centriole length and its intrinsic tubulin-dimer binding activity is required for procentriole elongation.

Functional characterization of the microtubule-binding and -destabilizing domains of CPAP and d-SAS-4.

We previously identified a novel centrosomal protein CPAP, which carries a 112-residue motif that is essential for microtubule destabilization. In this report, we define both the microtubule (MT) binding and destabilizing domains in human CPAP and analyze the mutations that affect its MT-destabilizing activity. Analysis of a series of CPAP truncated proteins showed that the MT-binding domain (MBD; residues 423-607) of CPAP is located next to its MT-destabilizing domain (MDD; residues 311-422). Site-specific mutagenesis revealed that the mutations that either disrupt the alpha-helical structure (Y341P, I346P, L348P, and triple-P) or alter the charge property (KR377EE) of the MDD significantly affect its MT-destabilizing ability. The activity for binding to a tubulin heterodimer was also significantly reduced in KR377EE mutant. Furthermore, we have analyzed the putative function of Drosophila d-SAS-4, a distant relative of human CPAP, which shares a conserved approximately 20-aa sequence with the MDD of CPAP. Our results show that mutations in this conserved sequence also eliminate d-SAS-4's MT-destabilizing activity, suggesting that d-SAS-4 and CPAP may play similar roles within cells.

Detecting low concentrations of Shigella sonnei in environmental water samples by PCR.

Outbreaks of Shigella sonnei associated with contaminated water have been reported and methods for the simultaneous detection of Shigellae and enteroinvasive Escherichia coli in water samples have been developed with detection limits of 10(1)-10(2) CFU mL(-1) of water. Because 10(1)-10(2)Shigellae can cause disease, a more sensitive detection method as an addition to the existing methods for detection of Shigella sonnei in water samples is reported here. Initially, 33 Shigella sonnei and 72 non-Shigella sonnei isolates were tested and one primer pair was found capable of specifically amplifying a 369-bp insertion sequence 1 (IS1) fragment from all 33 Shigella sonnei isolates and one Shigella dysenteriae ATCC isolate by PCR. The detection method was developed, which included filtration of 50 mL of water through a membrane and application of PCR to the membrane using this primer pair. Environmental water samples with total bacterial numbers of 384-2.84 x 10(7) CFU L(-1) were collected and seeded with 13 Shigella sonnei and the Shigella dysenteriae ATCC isolates. Detection limits were determined as 1.7-24.7 and 270-8000 CFU per 50 mL of water, respectively, using this detection method.

Conversion of Shigella flexneri serotype 2a to serotype Y in a shigellosis patient due to a single amino acid substitution in the protein product of the bacterial glucosyltransferase gtrII gene.

Conversion of serotype from 2a to Y was demonstrated with five Shigella flexneri isolates recovered from an infected patient. When introduced into the serotype Y isolate, the glucosyltransferase (gtr) II gene of the serotype 2a isolate is capable of inducing the conversion from serotype Y to 2a. In contrast, the gtrII of the serotype Y isolate lacks the capacity to change serotype, resulting from a Cys-->Tyr substitution in its predicted protein sequence. The protein product of the gtrII gene was detected. This is the first report of serotype conversion of S. flexneri in humans, and successful detection of the protein product from a gtr gene.

Molecular epidemiology of Shigella in a Taiwan township during 1996 to 2000.

A previously identified Shigella flexneri serotype 2a strain was responsible for an outbreak of shigellosis in a Taiwan township in August 1996. In order to find the relationship between this outbreak strain and subsequent Shigella infections in the area, 59, 47, 35, and 20 Shigella isolates recovered in 1997, 1998, 1999, and 2000, respectively, were collected and typed by serological and pulsed-field gel electrophoresis (PFGE) techniques. Of these 161 isolates, 139 isolates were S. flexneri serotype 2a, and one-third of them (47 isolates) exhibited the outbreak pattern. The remaining 92 S. flexneri serotype 2a isolates displayed 49 different NotI-PFGE patterns. Forty-five patterns were closely related to the outbreak pattern, with deletions of three specific NotI fragments occurring with high frequency. While the outbreak strain remained the main cause of shigellosis after the outbreak, the continuous emergence of closely related though poorly transmissible strains from the outbreak strain contributed to the observed annual decrease of shigellosis in the area.

The IS1 elements in Shigella boydii: horizontal transfer, vertical inactivation and target duplication.

IS1(SB) and its two variants were identified as the major and minor IS1 elements in Shigella boydii. The nucleotide sequences of IS1(SB), IS1(O157:H7) from Escherichia coli O157:H7 and IS1F from E. coli K12 suggest that these IS1 elements had been horizontally transferred among S. boydii and E. coli O157:H7 and K12. The two IS1(SB) variants and IS1(O157:H7) have transposition activities 7- to 86-fold less than that of IS1(SB), whereas IS1F has little transposition activity. Analysis of the flanking sequences of IS1(SB) and its two variants in S. boydii revealed the nature of regional specificity of the target sites and the sequence dependence of 8 and 9 bp target duplications, for which a model is presented.