Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)

A novel signaling cascade controlling actin polymerization in response to extracellular signals regulates filopodia formation and likely also neuronal synapse formation

Silencing of Exo70 leads to Cav1 accumulation in focal adhesions.

<p>Mock-treated (<b>A</b>) or Exo70-depleted cells (<b>B</b>) were detached for 1 h and replated on fibronectin-coated substrate for 3 h, and then fixed and stained for endogenous vinculin and Cav1. Scale bars, 5 µm. Co-localization between Cav1 and vinculin was quantified in a 20-pixel width region from the cell periphery and compared in Exo70-depleted with two independent siRNAs <i>vs</i>. mock-treated cells (<b>C</b>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052627#s2" target="_blank">Results</a> in panel C are the average of mean percentage of co-localization ± s.e.m. of three experiments. ** <i>P</i><0.05. Experiment has been repeated three times with number of cells analysed: n = 25 mock cells, n = 9 for cells treated with Exo70 siRNA 7c, and n = 23 for siRNA 7d.</p

Exo70 redistributes in Cav1-positive compartments upon cell detachment.

<p>(<b>A</b>) Hela cells expressing Cav1-mRFP and Exo70-GFP were kept in suspension for 1 h and replated on fibronectin for 3 h, and then visualized by confocal dual-colour spinning-disk microscopy (see corresponding movie S1). Arrow points to a dynamic Cav1- and Exo70-positive vesicle. Bottom panel shows selected frames from the time-lapse series corresponding to the boxed region in the upper panel. Time is given in second. (<b>B</b>) Hela cells expressing Cav1-GFP and cavin-1-mRFP were treated as in panel A. Inset shows higher magnification of region indicated by an arrow. Scale bars, 5 µm.</p

Actin and microtubule cytoskeletons act at distinct steps of Cav1 trafficking.

<p>(<b>A, B</b>) Hela cells expressing Cav1-mRFP maintained in suspension for 1 h, were replated on fibronectin for 3 h in the presence of 10 µM nocodazole (panel A) or 10 µg/ml cytochalasin-B (panel B) for 30 min. Cells were then analyzed by time-lapse confocal spinning disk microscopy. The right panels represent selected frames from the time-lapse series (time is given in second). Arrows point to Cav1-positive intracellular vesicles. See corresponding movie S2 (panel A) and movie 3 (panel B). Scale bars, 5 µm.</p