latrunculin-b and phosphatidylinositol-4-phosphate

Aspergillus nidulans hyphae grow exclusively by apical extension. Golgi equivalents (GEs) labeled with mRFP-tagged PH(OSBP) domain form a markedly polarized, dynamic network of ring-shaped and fenestrated cisternae that remains intact during "closed" mitosis. mRFP-PH(OSBP) GEs advance associated with the growing apex where secretion predominates but do not undergo long-distance movement toward the tip that could account for their polarization. mRFP-PH(OSBP) GEs overlap with the trans-Golgi resident Sec7 but do not colocalize with also polarized accretions of the early Golgi marker GrhA(Grh1)-GFP, indicating that early and late Golgi membranes segregate spatially. AnSec23-GFP ER exit sites (ERES) are numerous, relatively static foci localizing across the entire cell. However, their density is greatest near the tip, correlating with predominance of early and trans-Golgi elements in this region. Whereas GrhA-GFP structures and ERES reach the apical dome, mRFP-PH(OSBP) GEs are excluded from this region, which contains the endosome dynein loading zone. After latrunculin-mediated F-actin disruption, mRFP-PH(OSBP) GEs fragment and, like AnSec23-GFP ERES, depolarize. Brefeldin A transiently collapses late and early GEs into distinct aggregates containing Sec7/mRFP-PH(OSBP) and GrhA-GFP, respectively, temporarily arresting apical extension. Rapid growth reinitiates after washout, correlating with reacquisition of the normal Golgi organization that, we conclude, is required for apical extension.

Topics: Animals; Aspergillus nidulans; Brefeldin A; Bridged Bicyclo Compounds, Heterocyclic; Cell Polarity; Cell Shape; Fungal Proteins; Genes, Reporter; Genes, Synthetic; Golgi Apparatus; Humans; Hyphae; Image Processing, Computer-Assisted; Intracellular Membranes; Microscopy, Fluorescence; Mitosis; Organelles; Phosphatidylinositol Phosphates; Protein Structure, Tertiary; Rats; Receptors, Steroid; Recombinant Fusion Proteins; Thiazolidines; Type C Phospholipases