Departamento de Microbiología, Centro de Investigación Científica y de Educación Superior de Ensenada, Ensenada, Baja California, México.
PLoS One. 2013 Aug 12;8(8):e70655. doi: 10.1371/journal.pone.0070655. eCollection 2013.
The microtubule (MT) "plus end" constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called "MT plus-end tracking proteins" (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassaMicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.
微管（MT）“正端”构成了一组结构和功能多样的蛋白质的积累平台，统称为“MT 正端追踪蛋白”（+TIPs）。+TIPs 控制 MT 动态并将 MT 与多种亚细胞结构连接起来。粗糙脉孢菌微管结合蛋白-3（MTB-3）是酵母 EB1 的同源物，EB1 是一种高度保守的+TIP。为了研究 MTB-3 的功能，我们检查了 mtb-3 缺失的菌株，并通过 GFP 标记 MTB-3 来评估其动态行为。MTB-3-GFP 以彗星状结构存在，在菌丝细胞质中或多或少均匀分布，并以高达 4 倍于正常菌丝伸长率的速度主要向顶端移动。MTB-3-GFP 彗星存在于所有发育阶段，但在成熟菌丝中最为丰富。MTB-3-GFP 彗星观察到沿菌丝以顺行和逆行方向移动。逆行运动也观察到源自顶端圆顶。微管细胞骨架的完整性影响 MTB-3-GFP 彗星的存在和动态，而肌动蛋白似乎不起作用。MTB-3-GFP 彗星的大小受动力蛋白和传统驱动蛋白缺失的影响。我们在Δmtb-3 突变体中没有检测到明显的形态表型，但在Δmtb-3 中 MT 较少，MT 捆绑较少，组织较少。与 WT 相比，Δmtb-3 中的 MT 聚合和去聚合速率都显著降低。总之，缺乏 MTB-3 对粗糙脉孢菌的整体生长和形态表型影响很小，但 mtb-3 的缺失对 MT 动态有很强的影响。
