Dynamic localization of the Na+-HCO3- co-transporter NBCn1 to the plasma membrane, centrosomes, spindle and primary cilia.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal ß-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression.

Structural and Functional Analyses of the FAM46C/Plk4 Complex.

FAM46C, a non-canonical poly(A) polymerase, is frequently mutated in multiple myeloma. Loss of function of FAM46C promotes cell survival of multiple myeloma, suggesting a tumor-suppressive role. FAM46C is also essential for fastening sperm head and flagellum, indispensable for male fertility. The molecular mechanisms of these functions of FAM46C remain elusive. We report the crystal structure of FAM46C to provide the basis for its poly(A) polymerase activity and rationalize mutations associated with multiple myeloma. In addition, we found that FAM46C interacts directly with the serine/threonine kinase Plk4, the master regulator of centrosome duplication. We present the structure of FAM46C in complex with the Cryptic Polo-Box 1-2 domains of Plk4. Our structure-based mutational analyses show that the interaction with Plk4 recruits FAM46C to centrosomes. Our data suggest that Plk4-mediated localization of FAM46C enables its regulation of centrosome structure and functions, which may underlie the roles for FAM46C in cell proliferation and sperm development.

Subcellular drug targeting illuminates local kinase action.

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

Tryptone-stabilized gold nanoparticles induce unipolar clustering of supernumerary centrosomes and G1 arrest in triple-negative breast cancer cells.

Gold nanoparticles of different sizes, shapes, and decorations exert a variety of effects on biological systems. We report a novel mechanism of action of chemically modified, tryptone-stabilized gold nanoparticles (T-GNPs) in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231. The T-GNPs, synthesized using HAuCl4.3H2O and tryptone and characterized by an assortment of spectroscopy techniques combined with high-resolution electron microscopy, demonstrated strong antiproliferative and anti-clonogenic potential against MDA-MB-231 cells, arresting them at the G1 phase of the cell cycle and promoting apoptosis. The molecular mechanism of action of these particles involved induction of unipolar clustering and hyper amplification of the supernumerary centrosomes (a distinctive feature of many tumour cells, including TNBC cells). The clustering was facilitated by microtubules with suppressed dynamicity. Mass spectrometry-assisted proteomic analysis revealed that the T-GNP-induced G1 arrest was facilitated, at least in part, by downregulation of ribosome biogenesis pathways. Due to the presence of supernumerary centrosomes in many types of tumour cells, we propose chemical induction of their unipolar clustering as a potential therapeutic strategy.

Mid1ip1b modulates apical reorientation of non-centrosomal microtubule organizing center in epithelial cells.

In most kinds of animal cells, the centrosome serves as the main microtubule organizing center (MTOC) that nucleates microtubule arrays throughout the cytoplasm to maintain cell structure, cell division and intracellular transport. Whereas in epithelial cells, non-centrosomal MTOCs are established in the apical domain for generating asymmetric microtubule fibers and cilia in epithelial cells for the organ morphogenesis during embryonic development. However, the mechanism by which MTOCs localize to the apical domain in epithelial cells remains largely unknown. Here, we show that Mid1ip1b has a close interaction with γ-tubulin protein, the central component of MTOC, and modulates lumen opening of the neural tube, gut, intestine, and kidney of zebrafish. Knockdown or dominant negative effect of Mid1ip1b resulted in failure of lumen formation of the organs as aforementioned. Moreover, the non-centrosomal MTOCs were unable to orientate to the apical domain in Mid1ip1b knockdown epithelial cells, and the centrosomal MTOCs were inaccurately placed in the apical domain, resulting in defective formation of asymmetric microtubules and misplacement of cilia in the apical domain. These data uncover a molecule that controls the proper localization of MTOCs in the apical domain in epithelial cells for organ morphogenesis during embryonic development.

Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion.

Centrosome aberrations disrupt tissue architecture and may confer invasive properties to cancer cells. Here we show that structural centrosome aberrations, induced by overexpression of either Ninein-like protein (NLP) or CEP131/AZI1, sensitize polarized mammalian epithelia to basal cell extrusion. While unperturbed epithelia typically dispose of damaged cells through apical dissemination into luminal cavities, certain oncogenic mutations cause a switch in directionality towards basal cell extrusion, raising the potential for metastatic cell dissemination. Here we report that NLP-induced centrosome aberrations trigger the preferential extrusion of damaged cells towards the basal surface of epithelial monolayers. This switch in directionality from apical to basal dissemination coincides with a profound reorganization of the microtubule cytoskeleton, which in turn prevents the contractile ring repositioning that is required to support extrusion towards the apical surface. While the basal extrusion of cells harbouring NLP-induced centrosome aberrations requires exogenously induced cell damage, structural centrosome aberrations induced by excess CEP131 trigger the spontaneous dissemination of dying cells towards the basal surface from MDCK cysts. Thus, similar to oncogenic mutations, structural centrosome aberrations can favour basal extrusion of damaged cells from polarized epithelia. Assuming that additional mutations may promote cell survival, this process could sensitize epithelia to disseminate potentially metastatic cells.

The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome.

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.

Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration.

Cell migration, a complex process critical for tumor progression and metastasis, requires a dynamic crosstalk between microtubules (MTs) and focal adhesions (FAs). However, the molecular mechanisms underlying this event remain elusive. Herein we identify the proto-oncogenic protein Src as an important player in the regulation of the MT-FA crosstalk. Src interacts with and phosphorylates end-binding protein 1 (EB1), a member of MT plus end-tracking proteins (+TIPs), both in cells and in vitro. Systematic mutagenesis reveals that tyrosine-247 (Y247) is the primary residue of EB1 phosphorylated by Src. Interestingly, both constitutively activated Src and Y247-phosphorylated EB1 localize to the centrosome and FAs. Src-mediated EB1 phosphorylation diminishes its interactions with other +TIPs, including adenomatous polyposis coli (APC) and mitotic centromere associated kinesin (MCAK). In addition, EB1 phosphorylation at Y247 enhances the rate of MT catastrophe and significantly stimulates cell migration. These findings thus demonstrate that the Src-EB1 axis plays a crucial role in regulating the crosstalk between MTs and FAs to promote cell migration.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging.

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas-6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP-tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas-6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

Development and validation of a novel assay to identify radiosensitizers that target nucleophosmin 1.

A series of indole analogs that are synthesized using the scaffold of a potent radiosensitizer, YTR107, were tested for their ability to alter the solubility of phosphorylated nucleophosmin 1 (pNPM1). NPM1 is critical for DNA double strand break (DSB) repair. In response to formation of DNA DSBs, phosphorylated T199 NPM1 binds to ubiquitinated chromatin, in a RNF8/RNF168-dependent manner, forming irradiation-induced foci (IRIF) that promote repair of DNA DSBs. A Western blot assay was developed using lead molecule, YTR107, for the purpose of screening newly synthesized molecules that target pNPM1 in irradiated cells. A colony formation assay was used to demonstrate the radiosensitization properties of the compounds. Compounds that enhanced the extractability of pNPM1 upon radiation treatment possessed radiosensitization properties.

TACC3 protein regulates microtubule nucleation by affecting γ-tubulin ring complexes.

Centrosome-mediated microtubule nucleation is essential for spindle assembly during mitosis. Although γ-tubulin complexes have primarily been implicated in the nucleation process, details of the underlying mechanisms remain poorly understood. Here, we demonstrated that a member of the human transforming acidic coiled-coil (TACC) protein family, TACC3, plays a critical role in microtubule nucleation at the centrosome. In mitotic cells, TACC3 knockdown substantially affected the assembly of microtubules in the astral region and impaired microtubule nucleation at the centrosomes. The TACC3 depletion-induced mitotic phenotype was rescued by expression of the TACC3 C terminus predominantly consisting of the TACC domain, suggesting that the TACC domain plays an important role in microtubule assembly. Consistently, experiments with the recombinant TACC domain of TACC3 demonstrated that this domain possesses intrinsic microtubule nucleating activity. Co-immunoprecipitation and sedimentation experiments revealed that TACC3 mediates interactions with proteins of both the γ-tubulin ring complex (γ-TuRC) and the γ-tubulin small complex (γ-TuSC). Interestingly, TACC3 depletion resulted in reduced levels of γ-TuRC and increased levels of γ-TuSC, indicating that the assembly of γ-TuRC from γ-TuSC requires TACC3. Detailed analyses suggested that TACC3 facilitates the association of γ-TuSC-specific proteins with the proteins known to be involved in the assembly of γ-TuRC. Consistent with such a role for TACC3, the suppression of TACC3 disrupted localization of γ-TuRC proteins to the centrosome. Our findings reveal that TACC3 is involved in the regulation of microtubule nucleation at the centrosome and functions in the stabilization of the γ-tubulin ring complex assembly.

Reconstitution of cortical Dynein function.

Cytoplasmic dynein is a major microtubule (MT)-associated motor in nearly all eukaryotic cells. A subpopulation of dyneins associates with the cell cortex and the interaction of this cortical dynein with MTs helps to drive processes such as nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing. In this chapter, we describe three types of assays in which interactions between cortical dynein and MTs are reconstituted in vitro at increasing levels of complexity. In the first 1D assay, MTs, nucleated from a centrosome attached to a surface, grow against dynein-coated gold barriers. In this assay configuration, the interactions between MTs and dynein attached to a barrier can be studied in great detail. In the second and third assays, a freely moving dynamic aster is placed in either a 2D microfabricated chamber or a 3D water-in-oil emulsion droplet, with dynein-coated boundaries. These assays can be used to study how cortical dynein positions centrosomes. Finally, we discuss future possibilities for increasing the complexity of these reconstituted systems.

NFκB regulates expression of Polo-like kinase 4.

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulated NFκB activity in cancer can lead to increased genomic instability and uncontrolled proliferation.

Role of centrosomal adaptor proteins of the TACC family in the regulation of microtubule dynamics during mitotic cell division.

During the mitotic division cycle, cells pass through an extensive microtubule rearrangement process where microtubules forming the mitotic spindle apparatus are dynamically instable. Several centrosomal- and microtubule-associated proteins are involved in the regulation of microtubule dynamics and stability during mitosis. Here, we focus on members of the transforming acidic coiled coil (TACC) family of centrosomal adaptor proteins, in particular TACC3, in which their subcellular localization at the mitotic spindle apparatus is controlled by Aurora-A kinase-mediated phosphorylation. At the effector level, several TACC-binding partners have been identified and characterized in greater detail, in particular, the microtubule polymerase XMAP215/ch-TOG/CKAP5 and clathrin heavy chain (CHC). We summarize the recent progress in the molecular understanding of these TACC3 protein complexes, which are crucial for proper mitotic spindle assembly and dynamics to prevent faulty cell division and aneuploidy. In this regard, the (patho)biological role of TACC3 in development and cancer will be discussed.

Expression of centrosomal tubulins associated with DNA ploidy in breast premalignant lesions and carcinoma.

The centrosome plays an essential role in chromosomal segregation during cell division. Centrosome dysfunction might lead to aneuploidy and chromosomal instability. Invasive breast tumors with centrosome amplification often show aneuploidy. Flow cytometry (FCM) was used to examine the aneuploidy rate in 30 cases of each of the following seven tissue types: normal breast tissue, usual ductal hyperplasia, atypical ductal hyperplasia, low-grade ductal carcinoma in situ, high-grade ductal carcinoma in situ, low-grade invasive ductal carcinoma, and high-grade invasive ductal carcinoma. Centrosomal α, γ-tubulin expression was examined by FCM immunofluorescence and compared between diploid and aneuploid cells. The aneuploidy rate was 0, 6.7%, 26.7%, 30.0%, 46.7%, 56.7%, and 86.7%, respectively, in the seven tissue types. The percentage of cells expressing α- and γ-tubulins was significantly different between the seven groups, and the positive rate of α- and γ-tubulin expression in ADH, DCIS and IDC was higher than that in NBT and UDH. The percentage of cells expressing α- and γ-tubulins in the diploid state was significantly lower than that in the aneuploid state. Expression of centrosomal α- and γ-tubulins seems to be associated with DNA ploidy in breast premalignant lesions and carcinoma during the progression of breast cancer.

Poly-ADP ribosylation of Miki by tankyrase-1 promotes centrosome maturation.

During prometaphase, dense microtubule nucleation sites at centrosomes form robust spindles that align chromosomes promptly. Failure of centrosome maturation leaves chromosomes scattered, as seen routinely in cancer cells, including myelodysplastic syndrome (MDS). We previously reported that the Miki (LOC253012) gene is frequently deleted in MDS patients, and that low levels of Miki are associated with abnormal mitosis. Here we demonstrate that Miki localizes to the Golgi apparatus and is poly(ADP-ribosyl)ated by tankyrase-1 during late G2 and prophase. PARsylated Miki then translocates to mitotic centrosomes and anchors CG-NAP, a large scaffold protein of the γ-tubulin ring complex. Due to impairment of microtubule aster formation, cells in which tankyrase-1, Miki, or CG-NAP expression is downregulated all show prometaphase disturbances, including scattered and lagging chromosomes. Our data suggest that PARsylation of Miki by tankyrase-1 is a key initial event promoting prometaphase.

End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers.

During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent "end-on" fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein's interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries.

Girdin locates in centrosome and midbody and plays an important role in cell division.

Girdin is a downstream effector of epidermal growth factor receptor (EGFR)-AKT and interacts with actin and microtubule. Increasing evidence confirmed that Girdin played an important role in cell migration. Here we report that Girdin also regulates cell division. Overexpression or suppression of Girdin leads to attenuated cell proliferation. Imaging of mitotic cells revealed that Girdin is located in the cell division apparatus such as centrosome and midbody. The sub-cellular localization of Girdin was dependent on the domains, which interacted with actin or microtubules. Overexpression of Girdin lead to increased centrosome splitting and amplification. In addition, data show that pAKT also locates in both the centrosome and midbody, indicating the regulating role of AKT in Girdin-mediated cell division. To elucidate the effect of Girdin on tumor growth in vivo, HeLa cells infected with retrovirus harboring either control or Girdin shRNAs were injected subcutaneously into the immunocompromised nude mice. Downregulation of Girdin by shRNA markedly inhibited the cell growth of subcutaneously transplanted tumors in nude mice. These data demonstrate that Girdin is important for efficient cell division. Taking our previous data into consideration, we speculate that Girdin regulates both cell division and cell migration through cytoskeletal molecules.

Centrosomal localization of cyclins E and A: structural similarities and functional differences.

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G1/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity.

BACKGROUND INFORMATION: CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin-dependent kinase)-cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. RESULTS: In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N- and C-terminal regions of CDC25B and requires CDC25B binding to its CDK-cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. CONCLUSIONS: Our results therefore suggest that CDC25B associates with a Ctn2-containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components.