Ssp4在果蠅感覺神經元之研究

Abstract

在果蠅變態的過程中，修剪(pruning)是一個神經系統重塑的機制之一，也是一 個不會導致細胞死亡而去移除特定的神經細胞突出的自我摧毀過程。只要神經修 剪的過程出現錯誤都會使得整個神經系統出現異常，因此去了解在神經修剪過程 中的分子機制將有利於我們了解神經修剪是如何進行。第四樹突型神經(Class IV da neuron)之樹突在果蠅變態的過程中產生大規模的修剪，所以是一個很好的研究 神經修剪的模式。 現在已知在樹突修剪的過程中，微管(microtubule)會產生不穩定的狀態，這樣 不穩定的狀態會導致樹突開始斷裂(severing)。但是目前對於微管在幼蟲期(larval stage)是如何維持其穩定性尚未有一個明確的答案。在我們的研究中，我們發現一 個會和微管蛋白負端(microtubule minus end)結合的蛋白：Ssp4，Ssp4 負責在神經 中維持微管的完整性。我們觀察到在ssp4 的突變會導致幼蟲期的樹突提早發生斷 裂的現象。但是ssp4 RNA 干擾(RNAi)或ssp4 缺失都沒有導致幼蟲樹突提早發生 斷裂。從這個結果中我們認為我們在ssp4 突變下觀察到的樹突提早斷裂並不是由 於Ssp4 的功能出現問題所造成的。另一方面，Ssp4 的過度表現則會阻斷在結蛹期 的神經樹突的修剪。 為了更加了解Ssp4 的表現量是如何被調控的，我們研究了一個會和Ssp4 結合 的蛋白：Fem-1。目前知道線蟲的FEM-1 會作為Cul-2 泛素連接脢的受質轉接器 (substrate adaptor)調控蛋白質的降解。在S2 細胞中若大量表現Fem-1 會降地Ssp4 蛋白表現量，而在加入Cullin 抑制劑 MLN4924後則可以阻止Ssp4表現量的降低。 除此之外，我們也發現到Fem-1 會和Cul-2 和Cul-5 結合，暗示著Fem-1 可以做為 Cul-2 和Cul-5 的受質結合子。但當我們在細胞中特定的去降低Cul-2, Cul-4 或Cul-5 的表現量時，並不會導致Ssp4 的蛋白表現量有累積的情形，這代表Ssp4 的降解並 不是專門透過這三種Cullins。最後我們觀察到fem-1 突變以及ssp4 RNAi 的神經都 iii 會發生修剪的缺失，代表Fem-1 及Ssp4 對於樹突修剪這個過程是重要的。因此之 後的研究將會著重在Fem-1 及Ssp4 是如何影響樹突修剪的過程。

Pruning, one of the neuronal remodeling mechanisms during metamorphosis in Drosophila, is a tightly controlled process to eliminate specific parts of neuronal processes without causing cell death. Any mis-regulation of pruning activity could cause devastating consequences to the nervous systems. Thus, it is important to understand the mechanisms that regulate pruning activity in neurons. The dendrite pruning of class IV dendritic arborization (da) neurons, a subset of peripheral sensory neurons in Drosophila, provides a good model system to study the molecular mechanisms of dendrite pruning. It was known that microtubule destabilization at the proximal dendrites is one of the cellular events observed in pupal neurons during dendrite pruning. Thus, uncharacterized mechanisms are required to maintain microtubule stability in the dendrites of larval neurons and to suppress precocious dendrite pruning. Here we identified a protein, Ssp4 (short spindle 4, also called Patronin), which binds to and stabilizes the microtubule minus ends in Drosophila S2 cells, playing a role in the maintenance of microtubule integrity in neurons. We observed precocious dendrite severing in the neurons of heterozygous ssp4 mutant larvae. However, the class IV da neurons with ssp4 RNAi or of ssp4 imprecise excision mutants did not show any v precocious dendrite severing phenotypes in larval neurons. It indicated that precocious dendrite severing observed in the larval neurons of heterozygous ssp4 mutants is caused by other unidentified mutation, not by impaired function of Ssp4. On the other hand, overexpression of Ssp4 in class IV da neurons blocked dendrite severing at pupal stage. To understand how the function of Ssp4 is regulated, we studied an Ssp4-interacting protein Fem-1. Fem-1 is an ankyrin-repeat containing protein. It is known that C. elegans FEM-1 acts as a substrate adaptor of CUL-2 ubiquitin ligase complex and mediates protein degradation. Overexpression of Fem-1 decreased the amount of Ssp4 in S2 cells, suggesting that Fem-1 promotes Ssp4 degradation. Blocking Cullin activation by the treatment of inhibitor MLN4924 increased the Ssp4 level in S2 cells, implying that Ssp4 degradation depends on Cullin. These results suggested that Ssp4 is regulated by degradation through a Cullin-RING ligase complex that contains Fem-1 as the substrate adaptor. Moreover, Fem-1 could associate with Cul-2 and Cul-5 in S2 cells. However, knockdown of either cullin or double knockdown Cul-2 and Cul-5 did not cause the accumulation of Ssp4. This suggested that Ssp4 degradation is not specifically mediated by Cul-2 or Cul-5-RING ligase complex. Finally, we observed pruning defects in neurons with fem-1 hypomorph mutant pupae and in neurons with ssp4 RNAi. It suggested both Fem-1 and Ssp4 are required for dendrite pruning. Further studies are required to elucidate how fem-1 and ssp4 function during dendrite pruning.