First published online 30 November 2004
doi: 10.1242/jcs.01578
Journal of Cell Science 117, 6485-6495 (2004)
Published by The Company of Biologists 2004
Compartmentalisation of the sperm plasma membrane: a FRAP, FLIP and SPFI analysis of putative diffusion barriers on the sperm head
Peter S. James1,
Conor Hennessy2,
Torunn Berge3 and
Roy Jones1,*
1 Laboratory of Molecular Signalling, Babraham Institute, Cambridge, CB2 4AT, UK
2 Bioinformatics Section, Babraham Institute, Cambridge, CB2 4AT, UK
3 Department of Pharmacology, University of Cambridge, Cambridge, CB2 1QJ, UK
View larger version (79K):
[in a new window]
|
Fig. 1. Boar spermatozoa stained with DiIC16 by back-exchange from BSA-DiIC16. Live spermatozoa (A,B,C and upper sperm in D) showed uniform fluorescence throughout the plasma membrane with varying numbers of small strongly fluorescent particles attached to the head region. Dead sperm (lower sperm in D) were typically heavily stained on the acrosomal region (see Fig. 3 for definitions of compartments within the sperm head region). Bar, 4 µm.
|
|
View larger version (71K):
[in a new window]
|
Fig. 2. FLIP analysis of DiIC16 diffusion in the plasma membrane overlying the sperm head. (A) Separate frames from a video sequence to illustrate repeated photobleaching of a  2.0 µm diameter area located at the tip of the anterior acrosome (broken circle, frame 46). Only four out of the six bleaches are shown (bleaches were in frames 45, 153, 233 and 326). With each successive bleach, the intensity of fluorescence over the whole head declines uniformly. (B) (i) line-profile analysis of fluorescence intensity along the x-y axis shown in A in the pre-bleach state (frame 43), immediately post-bleach (frame 46), and after 3 seconds' recovery (frame 151); (ii) same as in (i) except after four successive bleaches (recovery frame 405). Arrows at `a' and `b' mark the anterior and posterior positions of the equatorial segment. Note that fluorescence intensity is similar across the equatorial segment-postacrosome boundary. (C) Similar to A except the bleach beam is located over the postacrosome (broken circle in pre-bleach frame 20). There was no difference in fluorescence intensity along the c-d axis after six successive bleaches (frame 554), indicating free diffusion of the lipid between surface compartments. Bar, 2 µm.
|
|
View larger version (34K):
[in a new window]
|
Fig. 3. AFM imaging of boar sperm heads with attached particles of DiIC16. (A) Contact mode image to illustrate topographical features and extent of the anterior acrosome, equatorial segment and postacrosome. (B) Line-profile analysis (A-B) across two sperm heads with attached particles (labelled X and Y) on the anterior acrosomal plasma membrane. In each case a small peak, 200 nm diameter, is coincident with the position of the DiIC16 particle. Bars, 2 µm in A; 1 µm in B.
|
|
View larger version (75K):
[in a new window]
|
Fig. 4. Trajectories of DiIC16 particles diffusing over the anterior acrosome and equatorial segment compartments of nine representative spermatozoa. The trajectories are colour-coded (beginning with dark blue to light blue to dark green to light green to brown to red) and change every 8 seconds. Bar, 2 µm.
|
|
View larger version (37K):
[in a new window]
|
Fig. 5. (A) A detailed analysis of particle trajectories reveals jump sizes of varying lengths. Two representative examples are shown. (B) Individual jumps are plotted in sequence. (C) Frequency of jump sizes. The most frequent jump sizes are equivalent to one and two pixel points (one pixel = 60 nm).
|
|
View larger version (16K):
[in a new window]
|
Fig. 6. Calculated D values (x10-9 cm2/second) of three representative particles diffusing between the equatorial segment and anterior acrosomal plasma membranes. An approximate periodicity is apparent irrespective of the position of the particle, but there are no consistent differences between the two compartments.
|
|
View larger version (17K):
[in a new window]
|
Fig. 7. Mean square displacement analysis of the nine particle trajectories (A-I) shown in Fig. 4. The essentially linear nature of the initial trajectories indicates random motion. When a particle approaches the edge of the head (as in B) or the boundary with the postacrosome (as in H), it becomes non-random due to restricted movement in the x or y axis. This coincides with a succession of short jumps before the particle moves away from the boundary.
|
|
View larger version (19K):
[in a new window]
|
Fig. 8. Proposed model for diffusion of individual molecules versus large complexes on the sperm head. In scenario (a), a single molecule (lipid or protein) is freely diffusing between all compartments. In (b), the molecule is part of a large complex that is unable to traverse the boundary between the postacrosme and equatorial segment. This creates the impression of a barrier. In (c), the complex disassembles in the postacrosomal side of the barrier, single molecules diffuse across it and reassemble on the equatorial segment. They may reform into their original complex or join existing complexes, all of which continue to diffuse randomly.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2004
