spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/jcs.00180

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Silverman-Gavrila, L. B.
Right arrow Articles by Lew, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Silverman-Gavrila, L. B.
Right arrow Articles by Lew, R. R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

An IP3-activated Ca2+ channel regulates fungal tip growth

Lorelei B. Silverman-Gavrila and Roger R. Lew*

Biology Department, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada



View larger version (25K):

[in a new window]
  		Fig. 1. (A) Internal generation and maintenance of the Ca2+ gradient in growing hyphae of N. crassa. This model is based upon extensive screening of inhibitors of Ca2+ signaling and PLC effects on hyphal growth, morphology, cytoplasmic and vesicular Ca2+ gradients, as well as direct evidence of IP3-activated Ca2+ channels using the BLM technique. Treatment with the Ca2+ ATPase inhibitor cyclopiazonic acid inhibited growth (hyphal widening was observed) and increased cytoplasmic [Ca2+] behind the apex, consistent with Ca2+ sequestration into endoplasmic reticulum behind the growing apex (Silverman-Gavrila and Lew, 2001Go). Microinjection of IP3 receptor agonists (IP3 and Adenophostin A) behind the tip inhibited growth transiently, caused subapical branching, and affected the tip-high cytoplasmic Ca2+ gradient; these effects were not observed after microinjection of the biologically inactive L-IP3 (Silverman-Gavrila and Lew, 2001Go). IP3-induced subapical branching was similar to subapical branching induced by ionophoretic injection of Ca2+ (Silverman-Gavrila and Lew, 2000Go), suggesting that IP3-activated Ca2+ release occurs in growing hyphae. An inhibitor of the IP3 receptor, 2-APB inhibited hyphal elongation and dissipated the tip high cytoplasmic [Ca2+] gradient. Thus, tip-localized IP3 production due to a stretch-activated phospholipase C could activate vesicular Ca2+ channels at the growing apex to generate the tip-high [Ca2+] gradient. The Ca2+ would induce fusion of wall vesicles at the apex, before being sequestered behind the tip via the Ca2+ ATPase. (B) The BLM technique used to identify and measure IP3-activated Ca2 channel activity in membrane vesicles isolated. from N. crassa. Hyphae are first homogenized to release the endomembranes. Following a series of centrifugations, subcellular fractions of membranes are isolated. Vesicles are added to the cis-chamber and fused to the lipid bilayer formed across an aperture in a septum that separates two chambers: cis and trans. The electronics are configured as a high gain current to voltage converter capable of measuring picoAmpere currents through ion channels. With only one permeant ion, Ca2+, only Ca2+ channels will be observed. Agonist addition to the cis compartment would activate channels oriented with their ligand binding site facing the cis compartment.

 


View larger version (18K):

[in a new window]
  		Fig. 2. (A) Activation of the channel by IP3, but not by the biologically inactive enantiomer L-IP3. After the formation of a lipid bilayer membrane across the aperture of the bilayer chambers, vesicles are added to the cis chamber. Vesicle fusion events with the bilayer are monitored by observing the appearance of channel activity (a), in this case at an applied voltage of +60 mV. The cis solution (1 M KCl, 10 mM CaCl2, 200 mM MOPS/BTP, pH 7.2) is replaced with 1 mM EGTA, 200 mM MOPS/BTP, pH 7.2 to remove all permeant ions except Ca2+. Trans-solution remained the same (50 mM Ca(OH)2, 200 mM MOPS/BTP, pH 7.2). No spontaneous Ca2+ channel activity is present after wash out (+60 mV clamp) (b). To demonstrate the presence of IP3-activated Ca2+ channel activity, the receptor agonist D-IP3 is added to the cis chamber. Addition of IP3 induces Ca2+ channel activity, with a conductance of about 10 pS (+60 mV clamp) (c). Channel activity is not present after removal of D-IP3 by wash-out (+40 mV clamp) (d). Ca2+ channels are not activated by addition of L-IP3 (+40 mV clamp) (e). The level marked closed on current traces represents zero current. The bars represent the current in pA (vertical) and time in seconds (horizontal). (B) The small conductance Ca2+ channel is inhibited by 2-APB but not by TMB-8. IP3 activates a small conductance Ca2+ channel (+50 mV clamp) (a). TMB-8 (200 µM) does not inhibit the channel (+70 mV clamp) (b), but 2-APB (25 µM) does (+50 mV clamp) (c).

 


View larger version (32K):

[in a new window]
  		Fig. 3. (A) Heparin does not inhibit the small conductance channel. After vesicle fusion, no spontaneous Ca2+ channels are observed in the absence of IP3 (40 mV clamp) (a). D-IP3 activates Ca2+ channels (12 pS) (+40 mV clamp) (b). The competitive IP3 channel blocker heparin (50 µM) has no effect on channel activity even after a second treatment (40 mV clamp) (c).(B) Dantrolene does not inhibit the small conductance channel. Prior to IP3 addition, no spontaneous Ca2+ channels are present (+60 mV clamp) (a). IP3 opens a small conductance channel (11 pS) (+100 mV clamp) (b). Dantrolene does not inhibit the channel (+100 mV clamp) (c).

 


View larger version (29K):

[in a new window]
  		Fig. 4. (A) TMB-8 inhibits the IP3-activated large conductance Ca2+ channel. Channel activity signals vesicle fusion (+100 mV clamp) (a). No spontaneous Ca2+ channels are observed after wash out (+45 mV clamp) (b). IP3 activates a large conductance channel (41 pS) (+70 mV clamp) (c). TMB-8 (200 µM) inhibits channel activity (+60 mV clamp) (d). (B) Heparin inhibits the large conductance channel. Representative channel current recordings show Ca2+ channel activation in response to the application of IP3 (+50 mV clamp) (a). The channels are inhibited by heparin (50 mV clamp) (b). (C) Dantrolene inhibits completely the large conductance channel. IP3-activated Ca2+ channel (131 pS) (+80 mV clamp) (a). Inhibition by 100 µM dantrolene (+80 mV clamp) (b).

 


View larger version (28K):

[in a new window]
  		Fig. 5. The small conductance channel conducts Ca2+, but not Mg2+. After vesicle fusion, no spontaneous Ca2+ channels are present (+60 mV clamp) (a). After IP3 addition, the small conductance channel does not conduct Mg2+ (+60 mV clamp) (b), but does conduct Ca2+ (12 pS) (+60 mV clamp) (c). 2-APB inhibits completely the channel (+50 mV clamp) (d).

 


View larger version (19K):

[in a new window]
  		Fig. 6. Current voltage curves of IP3-activated Ca2+ channels in N. crassa membranes. Current-voltage measurements reveal two distinct Ca2+channels: a small conductance channel (circles) and a large conductance channel (triangles). The current is recorded at different membrane potentials, then the voltage dependence of channel current is used to obtain current-voltage relations. Conductance and reversal potential are determined by linear regression. The calculated Nernst potential for Ca2+ is -33 mV for 100 µM Ca(OH)2 (calculated activity of 96 µM) in trans and 1 mM CaCl2 (calculated activity 7.09 µM) in the cis compartment. Note the difference in scale for small (left) and large (right) conductance channels. In this example, the channel conductance determined from the slope of current-voltages curves was 13 pS for the small channel and 111 pS for the large channel. Amplitude versus voltage was linear and reversed near -25 mV for the small channel and -16 mV for the large channel. Compiled data are presented in Table 3.

 


View larger version (41K):

[in a new window]
  		Fig. 7. Effects of PLC inhibitors on N. crassa growth rates. (A) Neomycin treatment. (Upper panel) Control experiments for treatment with external added inhibitors show that solution exchange (BS was exchanged with BS) does not affect hyphal growth. Middle and lower panels illustrate growth rates of hyphae treated with neomycin (as marked). Neomycin inhibits hyphal growth, partially at 100 µM and completely at 400 µM. (B) 3-nitrocoumarin causes a dose dependent inhibition of hyphal elongation: complete at 40 µg/ml, very efficient at 20 µg/ml and slight at 4 µg/ml. (C) Growth inhibition with increasing concentration of U-73122. (Upper panel) Control experiments. No influence on growth rates is observed for hyphae treated with the inactive analog U-73343. (Middle and lower panel) U-73122 inhibits growth: efficiently at 10 µM and completely at 400 µM within 4 minutes after its addition. Thin lines show individual experiments. Thicker lines and symbols show means±s.e.

 


View larger version (72K):

[in a new window]
  		Fig. 8. Effects of PLC inhibitors on hyphal growth in N. crassa. (A) U-73122 (400 µM) causes complete inhibition. (B) Control compound U-73343 did not affect hyphal elongation and morphology. (C) 3-nitrocoumarin (20 µg/ml) inhibited hyphal growth. The hypha recovered and restarted growth after washing out the inhibitor at 10 minutes. Pictures were taken at the time shown. Time 0 represents the addition of the inhibitor. Bars, 10 µm.

 


View larger version (98K):

[in a new window]
  		Fig. 9. Effects of Ca2+ and PLC inhibitors on organellar Ca2+ imaged with chlortetracycline. In normal growing hyphae significant fluorescence is detected associated with vesicles at the tip. (A) During Ca2+ imaging typical response (reduction of fluorescence at the tip, and an increase behind) is observed after the addition of 25 µM 2-APB at time 0 (n=18). At the same time hyphal elongation stops. (B) Similar results are obtained after treatment with 10 µM U-73122 (n=15). (C) Following the treatment with 400 µM neomycin similar effects occur (n=9). (D) In hyphae treated with cyclopiazonic acid the tip high Ca2+ is dissipated, but no increase in fluorescence behind the tip is observed (n=15). Bars, 10 µm.

 


View larger version (105K):

[in a new window]
  		Fig. 10. Hyperbranching after washing out inhibitors of IP3 receptor and PLC. (A) Within 20 minutes of washing out 25 µM 2-APB, hyphal widening, which extended over 20-40 µm from the tip, and an apical hyperbranching phenotype was observed. (B) U-73122 (10 µM) wash-out also caused hyphal widening and multiple apical branches. The picture was taken 14 minutes after wash out. (C) Control hypha 16 minutes after wash out. Hyperbranching was not observed after wash-out of cyclopiazonic acid (not shown); in this case, hypha resumed normal hyphal elongation, similar to C. Bars, 10 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002