The role of lipid homeostasis in nuclear shape and function
脂质稳态在核形状和功能中的作用
基本信息
- 批准号:7593726
- 负责人:
- 金额:$ 37.71万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:AffectAgingAnabolismArchitectureBiological ModelsCandidate Disease GeneCell CycleCell DeathCell NucleolusCell NucleusCell SurvivalCellsChromatinDefectElectron MicroscopyElementsEndoplasmic ReticulumEukaryotaEukaryotic CellExhibitsFatty acid glycerol estersGenesGenetic ScreeningGrowthHomeostasisHomologous ProteinInduced MutationLaminsLeadLinkLipidsMalignant NeoplasmsMembraneMitosisMorphologyMutateMutationNuclearNuclear EnvelopeNuclear LaminNuclear StructureNucleolar ProteinsObesityPeripheralPhospholipidsPlayProcessProteinsRestRoleRole playing therapySaccharomycetalesShapesStructureTestingTissue DifferentiationYeastsbasecell typelipinemutantspindle pole body
项目摘要
Alterations in nuclear structure are associated with aging and cancer, and yet the functional link between nuclear architecture and nuclear functions is not well understood. We have been using budding yeast as a model system to study nuclear architecture. The yeast nucleus differs from that of higher eukaryotes in two aspects: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in cells of metazoans. There is also accumulating evidence to suggest that lamins contribute to various nuclear processes. (2) The yeast nuclear membrane remains intact throughout the cell cycle, unlike nuclear membrane of higher eukaryotes, which breaks down during mitosis. Nonetheless, in an earlier study from our lab (Campbell et al, 2006), we showed that the shape of the yeast nucleus is determined by three factors: the composition of the nuclear membrane, the shape of the chromatin, and the presence of an unidentified nuclear structure that tethers the nuclear membrane to the chromatin, akin to nuclear lamins of higher eukaryotes. Our previous study focused on a yeast strain in which the Spo7 protein was inactivated. Spo7 is a regulator of phospholipids synthesis; in its absence phospholipids levels increase, leading to the expansion of the endoplasmic reticulum (ER) and certain regions of the nucleus. In particular, we were able to show that only the membrane associated with the nucleolus (a sub-compartment of the nucleus) expands, whereas the rest of the nuclear membrane remains juxtaposed to the bulk of the chromatin. This led to the hypothesis that in yeast there is a nuclear tether that associates the nuclear membrane to the chromatin and resists membrane expansion when phospholipids levels increase. Based on this hypothesis we assume that inactivating this tether will further alter nuclear shape, to the point where nuclear functions will be severely compromised, and this will be reflected in reduced cell viability. Thus, we sought mutations whose combination with a spo7 mutation leads to cell death or severe growth defects. Two approaches were used to find such mutations: in the first we conducted a screen for randomly induced mutations that cause cell death in a yeast background lacking Spo7 function (known as a yeast synthetic lethal screen). This was followed by a secondary screen for mutants that have an altered nuclear shape. In a pilot screen we have identified two mutations that lead to an altered nuclear shape, one of which is in a nucleolar protein. We are currently expanding the screen to uncover more proteins that contribute to nuclear shape. Once identified, we will study how these proteins affect nuclear shape and what role they play in coordinating nuclear architecture with nuclear function.
In the second approach we combined mutations in candidate genes with the spo7 mutation, looking for reduced growth in the double mutant. The candidate genes were selected based on their function and localization; most were involved in processes that take place near or at the nuclear membrane and most exhibited a peripheral localization. Through this screen we found one mutant that showed a severe growth defect when combined with a spo7 mutation. This mutant was known to affect spindle pole body function, and therefore we tested additional mutations known to abrogate spindle pole body assembly. We found that in many cases the spindle pole body defect was exacerbated when Spo7 was also absent. These findings suggest that the composition of the nuclear membrane affects the function of associated structures. We are now collaborating with Dr. Sue Jaspersen in order to look at spindle pole bodies by electron microscopy and to determine the relationship between nuclear membrane composition and the assembly of integral membrane structures.
核结构的改变与衰老和癌症有关,但核结构和核功能之间的功能联系尚不清楚。我们一直使用发芽酵母作为研究核结构的模型系统。酵母核与高等真核生物的核在两个方面不同:(1)酵母缺乏层蛋白,层蛋白在后生动物细胞中对细胞核的形成起主要结构作用。也有越来越多的证据表明,板层对各种核过程都有贡献。(2)酵母核膜在整个细胞周期中保持完整,与高等真核生物的核膜不同,核膜在有丝分裂期间破裂。尽管如此,在我们实验室的早期研究中(Campbell等人,2006年),我们表明酵母核的形状由三个因素决定:核膜的组成,染色质的形状,以及是否存在一种将核膜与染色质捆绑在一起的未知核结构,类似于高等真核生物的核层。我们之前的研究集中在一株Spo7蛋白失活的酵母菌株上。Spo7是磷脂合成的调节剂;如果没有Spo7,磷脂水平会增加,导致内质网(ER)和细胞核的某些区域扩张。特别是,我们能够证明,只有与核仁相关的膜(核的一个亚室)扩张,而核膜的其余部分仍然与染色质的大部分并列。这导致了一种假设,即在酵母中,存在一种核系带,它将核膜与染色质联系在一起,并在磷脂水平增加时抵制核膜的扩张。基于这一假设,我们假设,使这种系链失活会进一步改变核的形状,以至于核功能将严重受损,这将反映在细胞存活率的降低上。因此,我们寻找与sp7突变相结合导致细胞死亡或严重生长缺陷的突变。两种方法被用来发现这样的突变:在第一种方法中,我们对在缺乏Spo7功能的酵母背景中导致细胞死亡的随机诱导突变进行了筛选(称为酵母合成致死筛选)。随后对核形状发生变化的突变体进行了二次筛查。在试点筛选中,我们已经确定了两种导致核形状改变的突变,其中一种是在核仁蛋白中。我们目前正在扩大筛查范围,以发现更多与核形状有关的蛋白质。一旦确定,我们将研究这些蛋白质如何影响核形状,以及它们在协调核结构和核功能方面发挥什么作用。
在第二种方法中,我们将候选基因的突变与sp7突变结合在一起,寻找双突变的生长减慢。候选基因是根据它们的功能和定位来选择的;大多数都参与了发生在核膜附近或在核膜上的过程,并且大多数都表现出外围定位。通过这个筛选,我们发现了一个突变体,当与sp7突变结合时,显示出严重的生长缺陷。已知该突变会影响纺锤体极体功能,因此我们测试了其他已知可破坏纺锤体极体组装的突变。我们发现,在许多情况下,当Spo7缺失时,纺锤体的极体缺陷会加剧。这些发现表明,核膜的组成影响相关结构的功能。我们现在正在与Sue Jaspersen博士合作,通过电子显微镜观察纺锤体极体,并确定核膜组成和完整膜结构组装之间的关系。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Orna Cohen-Fix其他文献
Orna Cohen-Fix的其他文献
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{{ truncateString('Orna Cohen-Fix', 18)}}的其他基金
PDS1, A REGULATOR OF MITOSIS IN BUDDING YEAST
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2173254 - 财政年份:1997
- 资助金额:
$ 37.71万 - 项目类别:
PDS1, A REGULATOR OF MITOSIS IN BUDDING YEAST
PDS1,芽殖酵母有丝分裂的调节因子
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2459278 - 财政年份:1997
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$ 37.71万 - 项目类别:
PDS1, A REGULATOR OF MITOSIS IN BUDDING YEAST
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