Hervás-Aguilar América, Peñalva Miguel A
Centro de Investigaciones Biológicas CSIC, Madrid, Spain.
Eukaryot Cell. 2010 Oct;9(10):1504-18. doi: 10.1128/EC.00119-10. Epub 2010 Aug 6.
The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical "comets" of AbpA.
构巢曲霉内吞内化蛋白SlaB至关重要，这与内吞作用在顶端延伸中的关键作用一致。我们通过基因替换构建了一个硝酸盐诱导、铵抑制的slaB1等位基因，用于条件性SlaB表达。视频显微镜显示，受抑制的slaB1细胞能够建立但无法维持稳定的极性轴，在最初正常的芽管出现后不久，以类似出芽酵母的形态停止生长。使用绿色荧光蛋白（GFP）标记的分泌型v-SNARE SynA，其在被有效内吞后持续循环回到质膜，我们确定SlaB对内吞作用至关重要，尽管它对于SynA以及t-SNARE Pep12分别向质膜和液泡膜的顺向运输是可有可无的。通过共聚焦显微镜观察，受抑制的slaB1芽管显示出深深的质膜内陷。铵到硝酸盐培养基的转换实验证明了极性维持缺失表型的可逆性，以及正常顶端延伸与SynA内吞恢复的相关性。相反，在芽管出现后已经生长很长时间的菌丝中下调SlaB会导致明显的极性维持缺陷，这与SynA内吞不足相关。因此，我们在此报告的内吞作用的消除与极性维持能力丧失之间的严格相关性支持了这样一种观点，即菌丝生长需要分泌和内吞作用的耦合。然而，下调slaB1的细胞形成了含有肌动蛋白结合蛋白AbpA的F-肌动蛋白团块，因此F-肌动蛋白调节异常作为顶端延伸缺陷的一个可能因素不能被完全忽视。用Latrunculin B处理下调SlaB的顶端减少了AbpA团块的形成，但没有促进生长，并揭示了AbpA皮质“彗星”的形成。
