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Rapid dynamics of the microtubule binding of ensconsin in vivo

¹ Departments of Biological Sciences, Anatomy & Cell Biology & Pathology, Columbia University, College of Physicians & Surgeons, 630 W. 168th St, Rm BB1213, New York, NY 10032-3702, USA
² Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA
³ Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA
⁴ Cell Biology and Institute for Childhood & Neglected Diseases, Scripps Research Institute, La Jolla, CA 92037, USA

View larger version (101K): [in a new window]   Fig. 1. Dynamics of GFP-ensconsin chimera speckles in vivo. Fluorescent speckle microscopy (FSM) was used to record the dynamics of 5xGFP-EMTB chimeras in living cells and cytoskeletons. (a-e) Time-lapse micrographs of 5xGFP-EMTB speckles in living cells. Arrows point to speckles that remain from one interval to the next, while arrowheads denote speckles that appear or disappear during the sequence. The bar in the right-hand corner of time-lapse series e shows the magnification of 1µm in all panels. (f-j) Kymographs of a single MT segment from the time-lapse sequences. As shown, a and f are from untreated, b and g, are from detergent-extracted, c and h are from ATP-reduced, d and i are from ATP-reduced and then recovered and e and j are from Taxol-treated cells. See Materials and Methods for details of the treatments.

View larger version (40K): [in a new window]   Fig. 2. Photobleaching and recovery of ensconsin chimera in untreated cells. (A) Time-lapse micrographs of the bleached area before (pre-bleach) at the time of bleaching (laser spot) and at intervals during the recovery. Elapsed time, in seconds, is shown in each micrograph. (B) Corrected fluorescence (total pixel intensity along a segment of a bleached MT) was plotted against time (in seconds) before and after each of two bleach events. The time of each bleach is marked with an arrow. Fluorescence shown has been corrected, using an unbleached segment of an adjacent MT, for decay caused by photobleaching during time-lapse imaging and cellular background has been subtracted. (C) Determination of kOFF. As described in Materials and Methods, the logarithm of the fraction of fluorescence recovered was plotted against time and the slope, kOFF, was determined by linear regression.

View larger version (28K): [in a new window]   Fig. 3. Photobleaching and recovery of ensconsin chimera in detergent-extracted cells. (A) Time-lapse micrographs of the bleached area are shown before (prebleach), at the time of bleaching (white circle, showing the position of the laser bleach, since no image was captured at the instant of the bleach) and at intervals during the recovery. Elapsed time, in seconds, is shown in each micrograph. (B) Corrected fluorescence of a bleached MT (pixel intensity) was plotted against time (in seconds) at intervals before and after bleaching. The time of the bleach is marked with an arrow; fluorescence shown has been corrected, using an unbleached segment of an adjacent MT, for decay caused by photobleaching during repeated illumination, and cellular background has been subtracted.

View larger version (55K): [in a new window]   Fig. 4. Measurement of bound and unbound 5xGFP-EMTB in living cells. In order to estimate KD,app, we quantified 5xGFP-EMTB bound to MTs and 5xGFP-EMTB free in the cytoplasm. Using soluble DS-red expressed in the same cells was used as a volume marker. As shown in the figure, images of GFP and DS-red were captured from the same cell. Next, in each image, the background fluorescence was subtracted from an area that contained MTs and regions free of MTs (shown in box). To quantify unbound 5xGFP-EMTB, paired regions that were devoid of microtubules were used to scale the DS-red image to obtain an image of unbound GFP. In the example shown, regions used were 1x2 µm in size. Bound 5xGFP-EMTB was calculated as (Total GFP) - (Bound GFP). Bound and unbound 5xGFP-EMTB were used to calculate the apparent dissociation constant, KD,app.

View larger version (64K): [in a new window]   Fig. 5. Dynamics of MAP interaction with MTs does not depend upon their state of post-translational detyrosination. Micrographs of fixed TC-7 cells show fluorescence of MAP chimera visualized with GFP(a,c,e,g) and immunofluorescence of detyrosinated tubulin (anti-Glu antibody) visualized with rhodamine secondary antibody(b,d,f,h). Cells were treated as follows: a,b, untreated; c,d, detergent-extracted; e,f, ATP-reduced (azide-treated); and g,h, Taxol-treated. Note that, although there are more detyrosinated MTs in treated cells than in untreated ones, the detyrosinated MTs constitute only a subset of the total array under all conditions. Bar, 10 µm.

View larger version (95K): [in a new window]   Fig. 6. In vivo MT-binding dynamics of GFP-ensconsin chimeras are reduced by treatment with staurosporine. Fluorescent speckle microscopy (FSM) was used to record the dynamics of 5xGFP-EMTB chimeras in living cells. (A,B,C) Time-lapse micrographs of 5xGFP-EMTB speckles in living cells, treated with (A) staurosporine (25 nM, 2 hours), a general inhibitor of protein kinases; (B) no treatment; and (C) sodium azide and deoxyglucose (5 mM and 1 mM, 30 minutes) to substantially reduce cellular ATP. Arrows indicate speckles that remain from one panel to the next; open, white arrowheads denote speckles that disappear during the time-lapse sequence; and black-filled arrowheads denote speckles that appear during the time-lapse sequence. Elapsed time of each exposure is shown in seconds under each panel. (D-F) Kymographs of a MT segment from each of the time-lapse sequences; the scale markers on the left indicate the distance and time over which the kymographs were made. Kymographs and micrographs are the same magnification. Note that treatment with staurosporine substantially reduced dynamics of 5xGFP-EMTB speckles (A,D), in comparison with untreated cells (B,E).

View larger version (17K): [in a new window]   Fig. 7. Kinase activity regulates ensconsin’s MT-binding dynamics in living cells. Two possible models by which kinase activity could affect the dynamics of ensconsin’s association with MTs in vivo. (A) The enzyme phosphorylation model involves a hypothetical enzyme that, when phosphorylated, promotes both ensconsin’s association with and dissociation from the MT. Note that this model predicts that ATP depletion or inhibition of protein kinases would bring about a decrease in ensconsin’s MT-binding dynamics, without a concomitant increase in MT-binding affinity. (B) The ensconsin phosphorylation model predicts that phosphorylation of ensconsin that occurs while it is bound to the MT enhances its dissociation, while dephosphorylation in the cytoplasm enhances its rebinding to the MT. This model would predict that ATP depletion or inhibition of protein kinases would decrease ensconsin’s MT-binding dynamics; an increase in MT-binding affinity of ensconsin would be expected to ensue unless the phosphatase shown was also dependent upon phosphorylation for its activity.

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