Regulation of mouse oocyte microtubule and organelle dynamics by PADI6 and the cytoplasmic lattices

Baker Institute for Animal Health, Cornell University, Ithaca, NY 14853, USA.
Developmental Biology (Impact Factor: 3.55). 02/2011; 350(2):311-22. DOI: 10.1016/j.ydbio.2010.11.033
Source: PubMed


Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.

Full-text preview

Available from:
  • Source
    • "It is possible that drug treatments alter peroxisome movement by mechanisms other than increasing microtubule stabilisation. For instance, increased peroxisome speeds may result from the reintegration of the microtubule lattice after drug treatment, in addition to the increased acetylated α-tubulin (Kan et al., 2011). A common feature of tubulin-binding compounds is a linkage to assembly, either the stabilization of a microtubule lattice by compounds like taxol or epothilone A, or the preferential formation of alternate lattice contacts and polymers at microtubule ends by compounds like colchicine (Amos, 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS) cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine) that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials.
    Biology Open 05/2014; 3(6). DOI:10.1242/bio.20147641 · 2.42 Impact Factor
  • Source
    • "Indirect immunofluorescence labeling confocal microscopy was undertaken as described previously [26]. Rabbit-anti-H4Cit3 (1:50, Abcam), rabbit-anti-H3Cit2 + 8 + 17 (1:50, Abcam), rabbit-anti-H3Cit26 (1:50, Abcam), rabbit-anti-hyper acetyl H4 (1:50, Millipore), mouse-anti-acetyl H3K9 (1:20, Abcam), rabbit-anti-acetyl H4K5 (1:50, Abcam), rabbit-anti-acetyl H3K18 (1:50, Abcam), and mouse-anti-dimethyl H3K9 (1:20, Abcam) antibodies were used for this study. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation.
    BMC Developmental Biology 06/2012; 12(1):19. DOI:10.1186/1471-213X-12-19 · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cell polarity and asymmetry play a fundamental role in embryo development. The unequal segregation of determinants, cues, and activities is the major event in the differentiation of cell fate and function in all multicellular organisms. In oocytes, polarity and asymmetry in the distribution of different molecules are prerequisites for the progression and proper outcome of embryonic development. The mouse oocyte, like the oocytes of other mammals, seems to apply a less stringent strategy of polarization than other vertebrates. The mouse embryo undergoes a regulative type of development, which permits the full rectification of development even if the embryo loses up to half of its cells or its size is experimentally doubled during the early stages of embryogenesis. Such pliability is strongly related to the proper oocyte polarization before fertilization. Thus, the molecular mechanisms leading to the development and maintenance of oocyte polarity must be included in any fundamental understanding of the principles of embryo development. In this chapter, we provide an overview of current knowledge regarding the development and maintenance of polarity and asymmetry in the distribution of organelles and molecules in the mouse oocyte. Curiously, the mouse oocyte becomes polarized at least twice during ontogenesis; the question of how this phenomenon is achieved and what role it might play is addressed in this chapter.
    Results and problems in cell differentiation 08/2012; 55:23-44. DOI:10.1007/978-3-642-30406-4_2
Show more