探討減數分裂時期酵母菌 Ssa3 蛋白與粗絲期檢控點之關係

Abstract

老鼠熱休克蛋白質 HSP70-2 (70-kDa Heat shock protein) 在減數分裂過程中扮演重要的角色，在酵母菌 (Saccharomyces cerevisiae) 中與老鼠 HSP70-2 最相似的是 Ssa3 (stress-seventy subfamily A) 蛋白質。在減數分裂前期，若染色體重組與聯會複合體 (synaptonemal complex, SC) 組成發生異常 (例如 zip1 突變株)，粗絲期檢控點 (pachytene checkpoint) 會讓細胞停滯在粗絲期，阻止核分裂的進行，直到缺失修復完成。先前本實驗室發現 SSA3 基因突變能使部分的 zip1 突變株略過粗絲期檢控點而繼續進行減數分裂，且 Ssa3 蛋白分布在粗絲期染色體上。本篇論文的主要目標在於進一步確定 Ssa3 蛋白質與粗絲期檢控點之間的關係。SSA3 基因突變會使部分zip1 突變株略過粗絲期檢控點，此隱抑 zip1 缺失的現象與酵母菌 PCH2 (pachytene checkpoint) 基因突變相似，不過 pch2 會完全地使 zip1 突變株略過粗絲期檢控點。此外，pch2 亦能使半數的重組缺失細胞 (dmc1 突變株) 略過粗絲期檢控點完成核分裂，但 ssa3 則無法隱抑 dmc1 的缺失，因此我們推測 Ssa3 可能參與在粗絲期檢控點的路徑，但僅針對 zip1 缺失所引發的粗絲期檢控點。先前研究顯示 Pch2 也分布於粗絲期染色體上，並參與在粗絲期檢控點的其中一條路徑，於是我們透過免疫螢光染色同時觀察 Ssa3 與 Pch2 在染色體上的位置，結果無論在野生型或 zip1 突變株中，兩者在染色體上的位置並無明顯的重疊，顯示 Ssa3 與 Pch2 可能分屬在不同條粗絲期檢控點的路徑。野生型與 zip1 突變株中，Ssa3 蛋白會沿著粗絲期染色體分布，在核仁位置有較累積的現象，但在 zip1 突變株中有觀察到偏向點狀的 Ssa3 分布。Red1 是染色體 axial element 組成蛋白之一，由 Ssa3 與 Red1 免疫螢光結果得知 Ssa3 可能位於 axial element 與聯會複合體 lateral element 上。因此，本文推測 Ssa3 蛋白可能有助於 axial elements/lateral elements 間的結合，進而穩定染色體或聯會複合體的構造，使粗絲期檢控點能被有效地活化與作用，將 zip1 缺失的細胞留滯在粗絲期。但 zip1 ssa3 突變株因缺少 Ssa3 使得染色體結構較不穩定，粗絲期檢控點的活化與功能受到影響，部分突變株因此能略過粗絲期，完成減數分裂產生孢子。 另一方面，pch2 突變株在 30℃ 下的產孢率與野生型相同，但高溫 32.5℃ 下 卻觀察到 pch2 突變株的產孢效能會發生嚴重的延遲與降低，於是進一步探討其原因。pch2 突變株的核分裂情形與產孢率會隨著培養溫度越高，而有越嚴重的延遲與降低現象，且細胞可能停滯在粗絲期階段。NDT80-bc 片段缺失突變會使細胞完全不受粗絲期檢控點之控制，透過觀察高溫下的 pch2 NDT80-bc 突變株，顯示高溫造成 pch2 細胞停在粗絲期是粗絲期檢控點的調控所致。此外，不具有 SC 的zip1 pch2 突變株在高溫下能夠完成減數分裂並產生孢子，而具有少量 SC 的 zip3 pch2 突變株卻又受到檢控點的調控，大多數細胞會停滯在粗絲期，無法完成減數分裂。由以上結果推論，也許高溫會導致 pch2 細胞中的 SC 結構發生異常，而異常的 SC 會發出比缺乏 SC 更嚴重的訊號給檢控點，因此高溫下會觀察到 pch2 突變株而非 zip1 pch2 突變株受到粗絲期檢控點的調控，無法完成減數分裂與產生孢子。

Mutants that confer defects in meiotic recombination and synapsis (e.g. zip1) will trigger the pachytene checkpoint to delay and arrest cells at the pachytene stage of meiotic prophase. Previous studies have found that null mutant of yeast heat-shock protein SSA3 can partially suppress the checkpoint-mediated arrest of zip1; moreover, Ssa3 protein is localized to pachytene chromosomes, suggesting Ssa3 may function in the pachytene checkpoint. Here we show that SSA3 is specifically required for the pachytene arrest of zip1, for ssa3 fails to suppress pachytene-arrest of dmc1 mutant, in which the recombination is defective. Pch2, a protein localized to pachytene chromosomes and nucleolus, has been believed that it is involved in the pachytene checkpoint pathway. Using surface-spreading analyses, Ssa3 does not colocalize with Pch2, suggesting that probably Ssa3 does not involved in the Pch2-dependent checkpoint pathway. Ssa3 localizes along the length of pachytene chromosomes, and accumulates in the nucleolus in both wild type and zip1; however, we note that Ssa3 is present in dotty form on pachytene chromosomes in some zip1 mutant. Double staining of Ssa3 and Red1, a prominent component of axial elements/lateral elements of synaptonemal complex (SC), demonstrates that Ssa3 might localize to axial elements/lateral elements of SC. We propose that Ssa3 may provide its chaperon activity for maintaining a proper chromosomal status which is necessary for efficient activation of the pachytene checkpoint in budding yeast. On the other hand, we note that sporulation frequency of pch2 is similar to wild type at 30℃, while it is significantly delayed and reduced at high temperature, 32.5℃, thus we try to investigate the reason. Due to lots of pch2 mutants are arrested at pachytene at high temperature, we use pch2 NDT80-bc mutant to determine whether the pachytene-arrest of pch2 is mediated by the pachytene checkpoint. NDT80-bc is a deletion mutation of NDT80, which completely bypasses the checkpoint. pch2 NDT80-bc displays a wild type-like level and kinetics of sporulation and nuclear division, indicating that high temperature-modulated arrest of pch2 is pachytene checkpoint-mediated. Unlike pch2 mutant, sporulation of zip1 pch2 would not be affected at 32.5℃; as a result, we speculate may be the presence of SC is important for the activation of the pachytene checkpoint in pch2 at high temperature. The zip3 pch2 mutant, which generates partial SCs, is used to compare with zip1 pch2, which generates none of SC. Sporulation frequency and nuclear division of zip3 pch2 is decreased at 32.5℃, suggesting that SCs is required for checkpoint induction in pch2 at high temperature. For these reasons, we propose that high temperature might make SC aberrant in pch2, and the checkpoint signal from aberrant SC is much significant than from absent SC, so pch2 is arrested by the pachytene checkpoint at elevated temperature.