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Fig. 3. NPCs form at boundaries between rough and smooth membranes. Sperm chromatin was preincubated with NEP-B and cytosol before addition of NEP-A. Assembling sperm pronuclei were isolated and prepared for feSEM microscopy. (a-c) Each micrograph shows examples of the morphology of fusing vesicles observed five minutes after addition of NEP-A. At very high frequency, membranes with dense concentrations of ribosomes (labelled `dense') can be seen to be joined to membranes with sparse concentrations of ribosomes (`sparse'), and these are interpreted as initial fusion boundaries between single NEP-A and NEP-B vesicles. NPCs (arrows) and NPC assembly intermediates (arrowheads) were observed at the boundaries between dense and sparse areas of membranes. Bars, 100 nm. (d) Quantification of the position of NPC assembly was performed by measuring the distance from the nearest fusion seam of >300 randomly selected NPC intermediates in three independent experiments. The graph shows the percentage of NPC intermediates that were either at a fusion seam, within 200 µm of a fusion seam or >200 µm from a fusion seam. Bars, ±s.e.m.
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