Using an old Netscape Navigator 
or Internet Explorer (v. 2 or earlier)?
No  – jump to Index
Yes? Then you'll have problems based on the following.
<FONT FACE="Symbol"           >&#97; </FONT> (a)
   should = alpha symbol  (a, image )
<FONT FACE="Wingdings"        >&#108;</FONT> (l)
   should = closed circle (l, image )
<FONT FACE="Wingdings" size=-1>&#161;</FONT> (¡)
   should = open circle   (¡, image )
<FONT FACE="Symbol"           >&#180;</FONT> (´)
   should be an X-like symbol, image .
   <FONT FACE="Monotype Sorts">&#53; </FONT>
   = 5 = 5, instead of × (&#215;).
(&#181;)µ = µ (true character - code not needed)
(&#177;)± = ± (true character - code not needed)
In MSIE must order SUP and FONT SIZE, e.g.,  
   <SUP><FONT SIZE=1>2+</FONT></SUP> (²⁺), even though 
   <FONT SIZE=1><SUP>2+</SUP></FONT> (²⁺) works in NN.

Can't read some figure titles? Or other view problems?
If this 550-wide red line

extends past the edge of your windows, you probably have your monitor set for something like a low resolution (high magnification) of 640x480. Unless you have a tiny monitor or vision problem, this setting is quite impractical/suboptimal. You can try the frameless version of this page, but I'd bet web surfing is generally a hassle for you at this setting, so I'd change the setting – and make sure you are at >256 colors, too (unless you have an antique computer system!). In Windows, do this by way of your Control Panel's (in your "Main" Group, or under "Settings" of your "Start" button) Display Settings. (Click your browser's "Back" button to get where you came from.)

[reset or go/return to frames]

CONTENTS/INDEX

(MT = microtubule)

Abstract. Cell Motil. Cytoskeleton 41:168-180, 1998. © 1998 Wiley-Liss, Inc.

Fig.1. Translational activity of EF-1a in vitro & SDS-PAGE, purified carrot EF-1a.

Fig.2. Binding curve for EF-1a to MTs.

Fig.3. MTs assembled ± carrot EF-1a at 1:20.

Table 1. MT dynamic instability ± EF-1a: elongation & shortening velocities, catastrophe & rescue frequencies.

Fig.4. Plots of dynamic instability for MTs assembled ± EF-1a.

Fig.5. Axoneme-mediated assembly of animal tubulin ±/+ EF-1a.

Fig.6. Ca²⁺/CaM modulation of EF-1a-induced stabilization of MTs.

Fig.7. MT length distributions (+EF-1a) on plus and minus axoneme ends and +Ca²⁺ or Ca²⁺/CaM.

Fig.8. MT length distribution evidence that EF-1a does not sever, then bundle, taxol-stabilized MTs.

Fig.9. Evidence that presumed MT-severing activity is attributable to the mounting medium MOWIOL, not EF-1a.

Contact webmaster = "Dr. NAD" = Neil A. Durso, second author.

^ top ^

<Drag border to re-size frame.

Cell Motil. Cytoskeleton, vol 41:168-180, 1998. Click here for copyright (©) information.

Elongation Factor-1a Stabilizes Microtubules in a Calcium/Calmodulin-Dependent Manner
Richard C. Moore, Neil A. Durso, and Richard J. Cyr
Department of Biology and Intercollege Program in Plant Physiology, Penn State University, University Park, Pennsylvania
Neil A. Durso: Laboratory of Drug Discovery Research and Development, National Cancer Institute, NIH, Bethesda, MD Abstract
Elongation factor-1a (EF-1a), a highly conserved protein named for its role in protein translation, is also a microtubule-associated protein (MAP). We used high-resolution differential interference contrast microscopy to quantify the effect of substoichiometric amounts of a (isolated from Daucus carota) on the dynamic instability of microtubules assembled in vitro from either animal or plant tubulin. EF-1a modulates the dynamic behavior of microtubules assembled from either tubulin source, resulting in longer and more persistent microtubules. EF-1a, at a 1:20 molar ratio to tubulin, significantly (P < 0.05) reduces the frequency of catastrophe threefold and decreases shortening velocities almost twofold for microtubules assembled from animal tubulin. For microtubules assembled from plant tubulin, substoichiometric amounts of EF-1a significantly (P < 0.05) suppress the frequency of catastrophe greater than twofold and causes an almost threefold reduction in shortening velocities. Elongation velocities increase almost twofold and rescues, which are not observed in the absence of EF-1a, occur. In addition, calcium/calmodulin (Ca²⁺/CaM), which regulates the ability of EF-1a to bundle taxol-stabilized microtubules in vitro, also modulates the effect of EF-1a on the dynamic behavior of microtubules assembled in vitro from animal tubulin. Microtubule severing in the presence of EF-1a was never observed. These data support the hypothesis that EF-1a modulates the dynamic behavior of microtubules assembled in vitro in a Ca²⁺/CaM-dependent manner. click here for copyright (©) information

^ top ^

Fig. 1. a: Assays of the translational activity of EF-1a, using an in vitro translation system reconstituted principally from rabbit reticulocyte lysate.: Because UUU is a codon for the amino acid, Phe, ribosomes programmed with RNA, poly[U], synthesize poly[Phe] from Phe presented by aminoacylated tRNA^Phe. Polymeric Phe can be separated from monomeric Phe by filtration and the quantity of poly[Phe] produced is plotted as a function of EF-1a present. Rabbit EF-1a was assayed as a system standard at pH 7.5 (filled circles, l), or after dialysis against a buffer (pH 6.9) more compatible with microtubule biochemistry, in which the carrot EF-1a was also prepared (open circles, ¡). The results for rabbit indicate that an EF-1a buffer pH of 6.9 appears to reduce activity only slightly. The carrot samples were enriched for EF-1a by DEAE and CM chromatography, and the indicated values (X's, ×) based on quantitative protein gel scans (n = 3, ± SEM). These results closely agree with those for the carrot EF-1a homologue, PIK-A49; hence, carrot EF-1a thus obtained is translationally active. In sum, these data indicate that carrot EF-1a so prepared for use in microtubule assembly experiments is also translationally active.
b: SDS-PAGE (12% gel) analysis of carrot EF-1a preparation stained with Coomassie Brilliant Blue R-250.

^ top ^

Fig. 2. Binding curve plotted as EF-1a bound versus EF-1a free at 10 µM taxol-stabilized microtubules as determined from cosedimentation analysis.: EF-1a binds tightly to taxol-stabilized microtubules, with an approximate Kd value of 0.25 µM. Data points represent averages of 9-12 co-sedimentation experiments with coefficients of variation (SD/Mean) between 0.05 and 0.20.

^ top ^

Fig. 3. Differential interference contrast images of microtubules assembled in vitro from either animal or plant tubulin in the absence or presence of carrot EF-1a at a 1:20 molar ratio of EF-1a to tubulin.: Microtubules assembled in vitro from 25 µM animal tubulin in the absence of EF-1a (a) increase in length and number when assembled in the presence of 1.25 µM EF-1a (b). Microtubules assembled in vitro from 60 µM plant tubulin in the absence of EF-1a (c) also increase in length in the presence of 3 µM EF-1a (d). Scale bar = 2 µm.

^ top ^

Table 1. Microtubule dynamic instability in the presence and absence of EF-1a*

(a-b) Microtubules assembled in vitro from 25 µM animal tubulin ± 1:20 EF-1a. (c-d) Microtubules assembled in vitro from 60 µM plant tubulin ± 1:20 EF-1a.

Kinetic Parameters	a 0 µM EF-1a	b 1.25 µM EF-1a	c 0 µM EF-1a	d 3 µM EF-1a

V_e (µm/min)	1.6 ± 0.5 (n=78)	1.8 ± 0.7 (n=221)	1.3 ± 0.5 (n=23)	2.0 ± 0.5 (n=33)^a
V_s (µm/min)	14.1 ± 4.5 (n=95)	7.8 ± 2.4 (n=148)^a	210.3 ± 54.9 (n=22)	76.1 ± 25.2 (n=20)^a
k_c (s^-1)	0.0045 (Total time, 90 min)	0.0015^b (Total time, 264.2 min)	0.025 (Total time, 27.3 min)	0.011^b (Total time; 64.4 min)
k_r (s^-1)	0.0055 (Total time, 12.1 min)	0.0043 (Total time; 42.3 min)	0 (Total time, 0.24 min)	0.052^b (Total time; 0.96 min)
Pause (%)	56.7 ± 18.8 (n=20)	44.4 ± 8.8 (n=20)^a	83.5 ± 23.0 (n=22)	66.9 ± 16.6 (n=20)^a
Length (µm)	8.6 ± 4.4 (n=20)	20.5 ± 6.5 (n=21)^a	2.1 ± 1.4 (n=22)	3.7 ± 1.7 (n=20)^a

V_e, elongation velocity; V_s, shortening velocity; k_c, frequency of catastrophe; k_r frequency of rescue a, b: Microtubules assembled in vitro from 25 µM animal tubulin ± 1:20 EF-1a. c, d: Microtubules assembled in vitro from 60 µM plant tubulin ± 1:20 EF-1a. ^a Significantly different from 0 µM EF-1a, P < 0.05, one-way ANOVA. ^b Significantly different from 0 µM EF-1a, P < 0.05, analysis of Poisson 95% confidence intervals.

^ top ^

Fig. 4. Representative plots of dynamic instability for microtubules (MT) assembled in vitro from either animal or plant tubulin in the absence or presence of carrot EF-1a at a 1:20 molar ratio of EF-1a to tubulin. (can't see whole figure title): Microtubules assembled in vitro from animal tubulin (25 µM) in the absence of EF-1a (a) experience catastrophes more frequently and shorten more quickly than microtubules assembled in the presence of EF-1a (1.25 µM) (b). Microtubules assembled from plant tubulin (60 µM) in the absence of EF-1a (c) also have an increased frequency of catastrophe and shortening velocity compared with microtubules assembled in the presence of EF-1a (3 µM) (d). Rescues, which were not observed in the absence of EF-1a in microtubules assembled from plant tubulin, occur in the presence of EF-1a. Thus, EF-1a, through modulation of microtubule dynamic instability, promotes the growth and persistence of microtubules assembled in vitro from either animal or plant tubulin.

^ top ^

Fig. 5. Axoneme-mediated assembly of animal tubulin in the absence of EF-1a (filled circles, l), or in the presence of a 1:10 (open circles, ¡) or 1:20 (X's, ×) molar ratio of EF-1a to tubulin.: The mean number of microtubules per axoneme end were plotted (±SEM, n = 45 for absence of EF-1a, n = 25 for 1:10 and 1:20 EF-1a). EF-1a lowers the critical concentration for assembly onto both plus (a) and minus (b) axoneme ends (the plus end of the axoneme has longer and more microtubules than the minus end). A 1:10 molar ratio of EF-1a to tubulin is more effective at promoting microtubule assembly onto axonemes than a 1:20 molar ratio.

^ top ^

Fig. 6. Ca²⁺/CaM modulation of EF-1a-induced stabilization of microtubules assembled in vitro from animal tubulin.: 20 µM animal tubulin assembled onto axonemes in the presence of 1 µM EF-1a forms very long microtubules which are resistant to 50 µM Ca²⁺ (a). Upon addition of 50 µM Ca²⁺ / 10 µM CaM the microtubules undergo rapid shortening. At 3 min after the addition of Ca²⁺/CaM, microtubule 2 is completely depolymerized, while 1, 3, and 4 have appreciably shortened (b). Scale bar = 2 µM.

^ top ^

Fig. 7. Length distributions of microtubules assembled in vitro onto plus (a-c) and minus (d-f) axoneme ends from 20 µM animal tubulin in the presence 1 µM EF-1a and treated with 50 µM Ca²⁺ or 50 µM Ca²⁺ / 10 µM CaM.: The majority of microtubules assembled in the presence of EF-1a onto the axoneme plus end are longer than 20 µm, the length of the video screen (n= 125) (a). Microtubule length is unaffected by 50 µM Ca²⁺ (n=209) (b); however, in the presence of 50 µM Ca²⁺ and 10 µM CaM, there is a dramatic shift in the microtubule length distribution, with most microtubules less than 10 µm in length (n=219) (c). Microtubules assembled onto axoneme minus ends in the presence 1:20 EF-1a are shorter and less numerous than microtubules assembled onto axoneme plus ends (n=50) (d) and are sensitive to 50 µM Ca²⁺ alone, even in the presence of EF-1a (n=36) (e). In the presence of 50 µM Ca²⁺ / 10 µM CaM, however, there is even further reduction in microtubule length distribution and number (n=14) (f).

^ top ^

Fig. 8. Length distributions of 2 µM taxol-stabilized microtubules assembled in vitro from animal tubulin before addition of 2 µM EF-1a and after ionic disruption of EF-1a induced microtubule bundles.: Two micromolar taxol-stabilized microtubules (n = 590) (a) bundle within 5 min of the addition of 2 µM EF-1a, although unbundled microtubules still remain. The lengths of these unbundled microtubules were measured (n = 360) (b). Noticeably missing are microtubules greater than 20 µm. Upon the addition of 150 mM NaCl, the bundles dissociate. The length distribution of the released microtubules (n = 450) (c), however, is not significantly different (one-way ANOVA, P>0.05) than before the addition of EF-1a, and microtubules greater than 20 µm are found. This suggests that EF-1a does not sever microtubules, then bundle the severed fragments. That is, evidence for any severing is altogether absent.

^ top ^

Fig. 9. Images of rhodamine-labeled, taxol-stabilized microtubules (2 µM) in the absence and presence of 2 µM EF-1a, before and after fixing with the mounting medium MOWIOL.: Rhodamine-labeled, taxol-stabilized microtubules (2 µM) (a) bundle in the presence of 2 µM EF-1a (b), but also when "fixed" with the mounting medium MOWIOL in the absence of EF-1a (c). When 2 µM taxol-stabilized microtubules previously incubated with 2 µM EF-1a are added to MOWIOL, small microtubule fragments can be seen (d) between and around large microtubule aggregates of 100-1,000 µM in length (e). a-d: Scale bar = 10 µm; e: scale bar = 50 µm.

^ top ^