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Microtubule dynamics were investigated in CHO and NRK cells by novel experimental approaches designed to evaluate the microtubule behavior in the cell interior. These approaches were: (1) laser photobleaching of a path through the centrosome; (2) direct observation of microtubules in centrosome-containing cytoplasts; (3) GFP-CLIP-170 expression as a marker for microtubule plus end growth; and (iv) sequential subtraction analysis. The combination of these approaches allowed us to obtain data where the density of microtubules had previously prevented conventional methods to be applicable.
In the steady state, nascent microtubules grew persistently from the centrosome towards the cell margin. Frequently, they arrived at the cell margin without undergoing any transition to the shortening phase. In contrast to the growth of microtubules, shortening of the plus ends from the periphery was non-persistent; that is, rescue was frequent and the extent of shortening showed a distribution of lengths reflecting a stochastic process. The combination of persistent growth and a cell boundary led to a difference in apparent microtubule behavior in the cell interior compared with that near the cell margin. Whereas microtubules in the cell interior showed asymmetric transition frequencies, their behavior near the cell margin showed frequent fluctuations between phases of shortening and growth. Complete microtubule turnover was accomplished by the relatively rare episodes of shortening back to the centrosome. Release from the centrosome with subsequent minus end shortening also occurred but was a minor mechanism for microtubule turnover compared with the plus end pathway.
We propose a life cycle for a microtubule which consists of rapid growth from the centrosome to the cell margin followed by an indefinite period of fluctuations of phases of shortening and growth. We suggest that persistent growth and asymmetric transition frequencies serve the biological function of providing a mechanism by which microtubules may rapidly accommodate to the changing shape and advancing edge of motile cells.
Rapid remodeling of the microtubule (MT) array is important in large motile cells because such cells are constantly in the process of changing their shape. In fibroblast-like cells, MTs form a radial array emerging from the centrosome with minus ends tethered at the centrosome and plus ends extending towards the cell periphery (reviewed by Desai and Mitchison, 1997; Keating and Borisy, 1999). Although many studies have dealt with aspects of MT dynamics, a full understanding of the MT life cycle has yet to be achieved. In particular, the first phases, namely formation at the centrosome and growth in the cell interior have not been analyzed directly because of technical limitations imposed by the high density of MTs in the centrosomal region.
In previous studies, the dynamics and turnover of MTs in cultured cell lines have been analyzed primarily using fluorescently labeled tubulin. The picture that has emerged from these studies is that MTs in vivo undergo frequent transitions between phases of growth and shortening ( Sammak et al., 1987; Cassimeris et al., 1988; Shelden and Wadsworth, 1993; Waterman-Storer and Salmon, 1997; Yvon and Wadsworth, 1997).
However, several considerations suggest that this picture is incomplete. One basic point is that almost all observations of individual MT dynamics in vivo have been made near the cell margin. This is because the cell is thinner and the MT density is lower near the edge of the cell, thus permitting better visualization of individual MTs. Consequently, MT dynamics in the cell interior have remained essentially unexplored and the formal possibility exists that they are different from that at the cell margin. A second point is that MT dynamics in vivo show complexity not seen in vitro. In vitro, MT dynamics are characterized by transitions between well defined phases of growth and shortening ( Mitchison and Kirschner, 1984a; Mitchison and Kirschner, 1984b; Horio and Hotani, 1986; Walker et al., 1988). In contrast, MTs in many cell types do not show well defined phases. In addition, MTs in vivo frequently are quiescent, neither growing nor shortening, a behavior termed pause. Growth and shortening are highly variable in terms of velocity, duration and extent but, in general, tend to be brief. For example, the mean growth length has been reported as 1.3μ m in PtK1 cells and 3.2 μm in CHO cells ( Shelden and Wadsworth, 1993).
The difficulty in clearly defining phases of growth and shortening in vivo led us ( Vorobjev et al., 1999; Vorobjev et al., 1997) to introduce an alternative description of MT dynamics. MT dynamics were considered as a 1-dimensional random walk of their plus ends along the cell radius and their overall properties were characterized by two parameters— a diffusion coefficient and a drift coefficient. The diffusion coefficient is a measure of the amplitude (squared) of growth and shortening excursions per unit time, while the drift parameter represents the imbalance of growth and shortening excursions over time. This framework provided an analysis of dynamics not dependent upon detailed assumptions of growth or shortening behavior. The diffusion and drift parameters permitted prediction of the steady state length distribution of MTs and the time for turnover of the MT population. However, turnover times predicted from reported dynamic instability parameters were substantially greater than experimental determinations ( Vorobjev et al., 1997). A similar conclusion was drawn from Monte Carlo analysis using the dynamic instability model ( Gliksman et al., 1993). Proceeding from the assumption that MT plus ends undergo stochastic excursions such as the random walk observed in the lamellar region of PtK1 cells ( Vorobjev et al., 1997) or in fish melanophores ( Vorobjev et al., 1999), the time for a nascent MT plus end starting from the centrosome to grow to the cell margin (or to shorten from the cell margin back to the centrosome) will be a few hours for a typical cultured cell of radius 25 μm. Such a long time seems incompatible with the requirements for rapid cytoskeletal remodeling in cell motility behavior. This disparity between theoretical and experimental analyses of MT turnover is a third point suggesting the incompleteness of our understanding of MT dynamics in vivo. Thus, we considered that the dynamics of MTs in the cell interior may somehow be different from that near the cell margin. Supporting this view, a few studies have reported the capacity of MTs in vivo to show behavior other than rapid fluctuations between shortening and growth. MTs have been observed to persistently grow into the lamellipodia of newt lung cells ( Waterman-Storer and Salmon, 1997) and to continuously elongate into newly formed protrusions of HGF-stimulated PtK1 cells ( Wadsworth, 1999).
These considerations taken together motivated us to reinvestigate the life cycle of MTs by procedures designed to evaluate their behavior in the cell interior and, in particular, in the vicinity of the centrosome. By employing a combination of novel approaches, we found that MT behavior in the cell interior indeed differed from that near the cell margin. Remarkably, nascent MTs freshly nucleated at the centrosome grew persistently until they approached the cell margin. Only then did they display the frequent fluctuations between growth and shortening that are considered to be the hallmark of dynamic instability. We suggest a revised view of MT dynamics in vivo in which the `default' condition of a nascent MT is persistent growth. In this view, `dynamic instability' at the cell margin results from the behavior of the MT system operating under the constraint of a `boundary condition'.
CHO-K1 and NRK cells were grown in F-10 medium supplemented with 10% fetal bovine serum and antibiotics on coverslips with photoetched locator grids (Bellco Glass, Vineland, NJ). For observation of MT dynamics in vivo, cells were cultured for 2 days after plating and were microinjected with Cy3-tagged tubulin at a needle concentration of 10 mg/ml. Cells were kept on the microscope stage at 36-37°C during observation and temperature was measured before and after each experiment. Cells injected with Cy3-tubulin were treated with the oxygen-depleting preparation, Oxyrase (Oxyrase, Ashland, OH), to reduce photodamage and photobleaching ( Mikhailov and Gundersen, 1995). Injected cells were observed on a Nikon Diaphot 300 inverted microscope equipped with a Plan 100×, 1.25 NA objective using a Cy3 filter set for observations of Cy3-labeled MTs and a GFP filter set for observation of GFP-CLIP-170 in transiently transfected cells. Images of 16-bit depth were collected with a CH350 slow scan, cooled CCD camera (Photometrics, Tucson, AZ) driven by Metamorph imaging software (Universal Imaging, Westchester, PA). The image was projected onto the CCD chip at a magnification of 250×, which corresponded to a resolution of 1 pixel=0.09 μm (11.1 pixels per μm). Time-lapse series of 50-200 images were collected at 3-5 second intervals. 16-bit images were processed and rescaled with Metamorph software and 8-bit images were prepared for presentation with Adobe PhotoShop (Adobe Systems, Mountain View, CA). To highlight MTs of interest in some figures, color overlays were painted with opacity of 15% within the color mode layer in Adobe PhotoShop.
Photobleaching was performed on a Zeiss IM-35 inverted microscope using a 3 W argon ion laser as described elsewhere ( Keating et al., 1997), except that the cells were incubated at 37°C. The laser beam was shaped into an approximately 20×3 μm bar using a cylindrical lens and a Neofluar 100×, 1.3 NA objective. The zone was placed to photobleach a path across a cell with the centrosome at its center.
Cytoplasts were prepared by a modification of a described method ( Karsenti et al., 1984). Briefly, 2 days after plating onto coverslips cells were treated with nocodazole (1 μg/ml) and cytochalasin D (1.5 μg/ml) for 90 minutes. Coverslips were then placed `cells-down' into centrifuge tubes containing culture medium with drugs and were centrifuged at 10,000 g for 25 minutes to enucleate the cells. Enucleation resulted in about equal numbers of cytoplasts containing or lacking the centrosome. Coverslips were washed with fresh medium to remove drugs and incubated for 2 hours for complete recovery of MTs in the cytoplasts. For observation of MTs, cells were microinjected with Cy3-tagged tubulin before enucleation. For simultaneous observation of MTs and CLIP-170 (see next section), cells were first transfected with and allowed to express GFP-CLIP-170, GFP-CLIP-170-positive cells were microinjected with Cy3-tubulin and then cytoplasts were prepared.
Cells were transfected by glass capillary microinjection of DNA into the nuclei following a previously described procedure ( Perez et al., 1999) with slight modification and were observed after 10-20 hours. Briefly, pCB6-myc-GFP-CLIP-170 was used at a needle concentration of 20 μg/ml, which gave sufficiently low expression levels of recombinant protein without addition of cycloheximide. For visualization of the centrosome in some experiments, HsCen-2-GFP DNA (human centrin) was mixed with pCB6-myc-GFP-CLIP-170 DNA that had the same promoter. The final needle concentration of each vector was the same, -20 μg/ml.
The position of active ends of MTs and the length of growth or shortening episodes were analyzed by subtraction of sequential images (In—In+1) in time-lapse series as described previously ( Vorobjev et al., 1999). The resultant difference images identified the extent of growth or shortening as black or white domains, respectively, at the ends of individual MTs. The threshold (minimal length) for determination of shortening or growth in subtraction analysis was set to 0.45 μm — that is, 5 pixels in the digital image. Lateral displacements of MTs were distinguished from growth and shortening episodes because they gave a parallel arrangement of long black and white segments, whereas growth or shortening gave single short segments that were either black or white but not both.
where si is displacement of a MT end between two sequential frames, sd is mean displacement, and Σ(ti) is total time of observation. Analysis of instantaneous displacement histograms is simple and gives an advantage that any MT whose plus end is visible in two consecutive frames is taken into account.
Mean velocity of MT growth was determined using direct observation of Cy3-labeled MTs and the CLIP-170 approach. For the direct observation approach, MTs were monitored until they reached 90% of cell radius. The velocity of apparent CLIP-170 movement was determined by tracking the head of the individual CLIP-170-positive structure from the centrosome until it disappeared within the cytoplasm or near the plasma membrane.
The extent of persistent growth was expressed both as an absolute distance and as a percentage of the local cell radius. For the latter calculation, the local cell radius was determined as the straight line distance from the centrosome to the cell margin in the direction of MT plus end or CLIP displacement.
Two problems have previously hindered the direct observation of MT nucleation at the centrosome. First, MT density at the centrosome is high, precluding visualization of individual MTs. Second, the cell is generally thick in the region of the centrosome, which results in substantial out-of-focus fluorescence that degrades imaging of individual MTs. To overcome these obstacles, we have employed two approaches. In the first approach, we photobleached a path across the centrosomal region to diminish the fluorescence density of pre-existing MTs. This permitted observation of nascent MTs growing from the centrosome in the direction of the path. In the second approach, we generated cytoplasts that, because they lack the nucleus, are much thinner than whole cells, thus facilitating direct observation of individual MTs while still close to the centrosome.
Cy3-tubulin was microinjected into cells and allowed to incorporate into MTs. The MTs in CHO cells are arranged in a predominantly radial pattern with the centrosome at its focus. An argon ion laser and an optical track with a cylindrical lens were used to photobleach a path across the cell with the centrosome in its center. MTs that were nucleated at the centrosome after the photobleaching and which grew in the direction of the path could then be visualized continuously beginning within seconds of their birth ( Fig. 1A).
Path photobleaching reveals persistent MT growth in the interior of a CHO cell. (A) Upper panels: low magnification images of Cy3-labeled MTs before and after bleaching a zone approximately 20×3 μm passing through the centrosomal region. The path of reduced fluorescence allows visualization of nascent centrosomal MTs. Lower panels: three MTs oriented along the direction of the path (colored yellow, red and green) can be seen to elongate persistently. Numbers in top-left corners indicate time in seconds. Images were acquired every 4 seconds. Bar, 5 μm. (B) Life history plots (length vs time) of colored MTs. Zero length represents position of the centrosome; zero time is time of bleaching. MT plus ends arrived at the plasma membrane in∼ 60 seconds. (C) Instantaneous velocities of nascent MTs were measured as the displacement of MT ends between successive frames. The histogram shows that episodes of shortening and pause were infrequent.
The nascent MTs displayed remarkable behavior not previously reported in animal cells. They showed negligible, if any, dynamic instability. Typically, their leading (presumably plus) ends grew persistently until they were at the cell periphery ( Fig. 1B). As an operational definition, we consider the cell periphery to be a zone of∼ 15% of the cell radius (3 μm). On average, 68% of the MTs reached the cell margin without transition to a shortening phase and continuous MT growth phases sometimes exceeded 20 μm. The growth rate derived from the frequency histogram of instantaneous rates ( Fig. 1C) was 17.8±13.8 μm/minute (n=33 MTs; 10 cells; total time of observation=1228 seconds). The histogram confirmed that episodes of shortening (8.4%) or pause (0.6%) comprised a minor fraction of nascent MT behavior. At the same time that nascent MTs were growing persistently in the interior, life history plots of MTs at the periphery of the cell showed that they displayed characteristic dynamic instability, both before and after photobleaching (data not shown). Therefore, the experimental protocol did not detectably affect MT behavior. Thus, observations after path photobleaching suggest that nascent MTs in the cell interior have different behavior than those at the periphery. However, a limitation of this approach was that only those MTs that were oriented along the path (usually 2-4 per cell) could be followed, limiting the size of the data set. To obtain a more comprehensive picture we examined cytoplasts where multiple MTs could be tracked starting close to the centrosome.
Enucleation of cells results in cytoplasts that are smaller and flatter than whole cells. The cytoplasts generally contained a reduced number of MTs (∼50%) compared with those in intact cells, suggesting that some nucleating material was lost or damaged during enucleation. Nevertheless, centrosome-containing cytoplasts retained a strong radial array of MTs with the centrosome at its center. The combination of thinness and reduced number of MTs allowed the centrosome to be clearly visible and MTs growing from it to be readily traceable ( Fig. 2A). Thus, it was possible to identify an individual MT shortly after it was nucleated and to monitor its elongation in cytoplasts without the need for path photobleaching. MTs grew with rare pauses or shortening episodes ( Fig. 2B) at a rate of 15.8±5.9 μm/minute (50 MTs; 8 cytoplasts), which was similar to that in whole cells after path photobleaching. Thus, the property of persistent MT growth was retained after enucleation.
Life cycle of MTs visualized in centrosome-containing CHO cytoplasts. (A) Time-lapse sequence shows selected panels from life histories of four MTs. (B) Two MTs (colored blue and red) were nucleated at the centrosome (at 3 seconds and 39 seconds, respectively) and persistently grew towards the cell margin. These MTs underwent transition to shortening only after they arrived at the cell margin (arrowheads: red MT, at 102 seconds; blue MT at 120 seconds). The length of the blue MT is greater than the red MT because it grew in a curved path and along the cell margin before beginning to shorten. (C) Two MTs (colored yellow and green) were initially tracked while near the cell margin. These MTs showed repeated episodes of growth and shortening near the membrane and the green MT eventually underwent a shortening excursion back to the centrosome. Numbers in the top-left corners indicate time in seconds. Bar, 5μ m. Life history plots constructed as indicated in Fig. 1 legend.
As a measure of MT persistence, we estimated the frequency of transition from growth to shortening. Times were recorded for the growth of nascent MTs from the moment they could be detected near the centrosome until they had a `catastrophe' (shortening episode >0.5 μm) or arrived at the cell periphery, whichever came first. For MTs that arrived at the cell periphery without a detectable shortening episode, we logged their times but did not log a catastrophe. Since MTs in whole cells and centrosome-containing cytoplasts behaved similarly, we combined data from both protocols. For 52 MTs analyzed, only 12 transitions to shortening were logged in 2337 seconds of observation time for a catastrophe frequency of 0.005 second-1.
As MTs approached the cell margin, their behavior changed. Continued observation of MTs for at least 3 minutes, starting from the centrosome ( Fig. 2B) or selecting them near the plasma membrane ( Fig. 2C) showed that the majority of plus ends displayed typical dynamic instability near the cell margin. MT behavior within the 3 μm zone from the cell margin was characterized by an apparent catastrophe frequency of 0.08 second-1 (207 events for 2710 seconds, 61 MTs analyzed in 17 cells and cytoplasts), that is, 16-fold higher than that in the cell interior. Thus, as in the path photobleaching experiments, dynamic instability behavior was observed only at the cell periphery.
The apparent difference in MT behavior in the cell interior as opposed to the cell periphery represents a significant departure from our understanding of MT dynamics. Therefore, it seemed advisable to seek confirmation of these conclusions by an independent approach. Ideally, one would want to visualize MT growing ends without the background contributed by the rest of the MTs. Such selective visualization seemed possible through the use of CLIP-170 as a marker for MT elongation ( Perez et al., 1999). Although Perez et al. showed that CLIP-170 co-localized with the ends of some growing MTs and was not targeted to stationary or shortening MTs, they were not able to determine whether CLIP-170 was targeted to all growing MTs or only to a certain subset. Thus, we first needed to evaluate this point. To do so, we compared MT growth with GFP-CLIP-170 behavior using a double labeling protocol for cytoplasts. Intact cells were transfected by microinjection of GFP-tagged CLIP-170 DNA into the nuclei. To avoid potential problems with overexpression, we selected cells at 10 hours after transfection expressing low levels of fluorescent GFP-CLIP-170. These GFP-positive cells were then injected with Cy3-labeled tubulin and incubated for 60-120 minutes prior to cytoplast preparation. Colocalization of GFP-CLIP-170 (red) and ends of MTs (green) in a centrosome-containing cytoplast is shown in Fig. 3A. Displacement of the CLIP-170 label in successive frames was identical to the growth of MT plus ends ( Fig. 3B). CLIP-170 labeling disappeared within 5 seconds (time resolution of our double channel time-lapse series, 2.5 seconds per channel) when a MT paused or underwent a transition from growth to shortening ( Fig. 3C). Several typical CLIP-170 and MT histories are plotted in Fig. 3D. Invariably (n>100), CLIP-170 tracks were coincident with MT tip displacement, thus indicating that CLIP-170 was targeted to all growing ends and validating their use as a tool for selectively visualizing MT growth.
Selective visualization of growing MT ends with CLIP-170. CHO cells were transfected by nuclear microinjection of GFP-CLIP-170 DNA, allowed to express GFP-CLIP protein, then microinjected with Cy3-tubulin and, finally, enucleated to generate cytoplasts. Time-lapse sequences were obtained to show dynamics of MTs and CLIP-170. (A) Low magnification of MTs, CLIP-170 and merged image (green, Cy3-MTs; red, CLIP-170). (B) Time-lapse sequence of region boxed in panel A. Two dynamic MTs are indicated by arrowheads. CLIP-170 is present at their plus ends during growth phases but disappears within 5 seconds after transition from growth to pause or shortening phase. Numbers in top-left corners indicate time in seconds. Interval between acquisitions of images in alternating channels was 2.5 seconds, which gave an interval between successive images in either the GFP or Cy3 channel of 5 seconds. Scale bar, 5μ m. (C) Life history plots of MTs (green) and CLIP-170 tracks (red) indicated by arrowheads in panel B. CLIP-170 data points were time-shifted by 2.5 seconds to compensate for the delay between acquisition of CLIP and MT images. Absence of red data points from segments of the plots indicates when CLIP-170 disappeared from the MT end. (D) Example plots of nascent MTs (green) growing off the centrosome and corresponding CLIP-170 tracks (red) after time-shifting of 2.5 s. Plots were arbitrarily staggered along the time axis for clarity. Scales of axes indicated in upper right corner of graph. For some MTs either near the centrosome or in areas of high MT density, the MT end could not be clearly visualized. Nevertheless, CLIP-170 movement could be seen. This is represented in the plots as red points without corresponding green ones. In all cases, clear growing MTs had CLIP-170 at their ends.
A prediction of persistent growth of MTs starting from the centrosome is that CLIP-170 will be associated with a MT tip almost continuously until the MT plus end approaches the cell margin. Therefore, we used the movement of CLIP-170 labeled zones as a tool to evaluate MT growth in the cell interior. For visualization of the centrosome in some experiments, GFP-Hscen2 DNA (human centrin expressing construct) was mixed with GFP-CLIP-170 DNA and they were used together for transfection.
We evaluated MT growth both in intact cells and in cytoplasts containing a centrosome. As predicted, GFP-CLIP-170-labeled zones displayed long excursions over time (continuous duration frequently >1 minute) through the cell interior ( Fig. 4A). Track diagrams were built from successive positions of CLIP-170-labeled zones on individual MTs. The tracks showed that CLIP-170 zones moved radially outward from the centrosome. The mean unbroken length of GFP-CLIP-170 tracks was 17.3±4.8 μm (n=78) in whole cells ( Fig. 4B) and 15.8±5.8μ m/minute (n=40) in cytoplasts containing a centrosome. The mean velocity of CLIP-170 zones was 16.5±6.0 μm/minute (n=110) for whole cells and 18.3±4.8 μm/minute (n=39) for the cytoplasts. The velocity of CLIP-170 movement was similar to that determined for MT growth by direct observation, both in whole cells and cytoplasts. We may conclude from these measurements both that CLIP-170 movement is a good proxy for MT growth and that cytoplasts are a good proxy for intact cells. Normalizing the length distribution of CLIP tracks against the distance between the centrosome and the cell margin ( Fig. 4C) shows that the majority of MTs nucleated at the centrosome reached ∼85% (84±16%) of the cell radius without catastrophes or long pauses. This result signifies that most MTs persistently grow from their time of birth at the centrosome until their plus end nears the cell margin.
Persistent MT growth confirmed by long CLIP-170 tracks. GFP-CLIP-170 movement was analyzed by time-lapse microscopy. Images were acquired every 3 seconds. (A) Three next-nearest frames are shown, displaying radial organization and movement of CLIP-170 patches. Tips of four CLIP-170 patches are indicated over time by arrowheads. Numbers in the upper left corner indicate time in seconds; bar, 5 μm. Diagram shows 25 CLIP-170 tracks over a 90 second period of which four tracks (1-4) correspond to CLIP patches indicated by arrowheads on the images. Many tracks are long indicating continuous MT growth; radial pattern of tracks permits identification of the centrosome region (dashed circle) as the point from which the tracks originate. The centrosome is indicated as two dots in the diagram. (B) Histogram of the length distribution of CLIP-170 tracks. Mean length, 17.3±4.8 μm (n=78). (C) Histogram of length distribution of CLIP tracks normalized against the distance between the centrosome and plasma membrane. The histogram shows that the majority of MTs born at the centrosome grow persistently to ∼85% of the cell radius.
Because new CLIP-170 labeled zones continuously appeared near the centrosome, it was possible to use their appearance to estimate the frequency of MT formation at the centrosome, defined operationally as a circle of 2μ m radius drawn about the focus of the MT array. With this criterion, we estimated the rate of nucleation of MTs by the centrosome in CHO cells to be 5.6±2.3 per minute (178 CLIP-170 zones; 7 cells; 1997 seconds).
The only previously reported instance of persistent plus end growth in mammalian cells is the MT treadmilling observed in vivo in the absence of a centrosome ( Rodionov and Borisy, 1997; Rodionov et al., 1999). In our previous study ( Rodionov et al., 1999) we suggested that treadmilling in cytoplasts lacking a centrosome resulted from exposed MT minus ends whose depolymerization caused an elevated tubulin pool that suppressed transitions from growth to shortening phase at the MT plus end. Contrary to that suggestion, this study has uncovered persistent growth of MT plus ends in the absence of substantial numbers of free minus ends. Therefore, we reevaluated the question of whether the presence of the centrosome had any significant effect on the rate of plus end growth. With our improved procedures, we compared cytoplasts containing and lacking the centrosome.
In centrosome-free cytoplasts, MTs spontaneously appeared in the cytoplasm, then translocated by means of treadmilling and eventually arrived at a cell margin ( Fig. 5A). After reaching the cell margin, plus ends often became paused and MTs disassembled because of minus end depolymerization. The rate of growth of the plus end during treadmilling was measured in the same way as for MTs growing from the centrosome in intact cells or centrosome-containing cytoplasts. From time-lapse observations of Cy3-labeled MTs, the rate of plus end growth during treadmilling was 20.3±8.3 μm/minute (n=66; 11 cytoplasts). In steady-state treadmilling, growth of the plus ends must be balanced by minus end shortening. Consistent with this expectation, the rate of minus end shortening in the cytoplasts lacking a centrosome was 18.2±5.8μ m/minute (n=66; 11 cytoplasts).
MT treadmilling and GFP-CLIP-170. MT dynamics and GFP-CLIP-170 movement were analysed in cytoplasts lacking the centrosome. (A) MTs and CLIP-170 images (two left pictures) and time lapse sequence of merged images with GFP-CLIP-170 set to the red channel and MTs to the green channel, respectively. Plus and minus ends of treadmilling MTs are indicated by arrowheads. CLIP-170 labels the plus ends of treadmilling MTs while minus ends are always CLIP-170-negative. Numbers in the top-left corner indicate time in seconds; bar, 5 μm. (B) Example histories of CLIP-170 tracks in cytoplasts lacking a centrosome. Individual plots are arbitrarily staggered along the time axis for clarity. Scale of axes is indicated in the top-right corner of the graph. (C) Life history plot of a treadmilling MT (plus and minus ends are green) and CLIP-170 (red). Time shift between plus end history and CLIP-170 history is 2.5 seconds. CLIP-170 persists at the growing plus end of the treadmilling MT.
Our measurement by direct observation of mean plus end growth rate includes pauses and shortening episodes. To compare the real growth potential of the plus ends these episodes have to be excluded. The CLIP-170 approach provides a natural realization of this algorithm since CLIP disappears from the plus ends when they stop. The tracks of CLIP-170 in cytoplasts lacking a centrosome ( Fig. 5B) gave the MT plus end growth rate as 22.0±10.1 μm/minute (n=75; 7 cytoplasts), while in centrosome-containing cytoplasts, the measured rate was 18.3±4.8 μm/minute (n=39; 5 cytoplasts), a difference of 20%. Thus, by both measures, MT growth was slightly faster in cytoplasts lacking a centrosome, consistent with an elevated tubulin pool. However, since persistent growth is a regular feature of MTs in intact cells and centrosome-containing cytoplasts, the creation of free minus ends is clearly not a prerequisite for this phenomenon.
Treadmilling MTs permitted an additional test of the localization of CLIP-170 exclusively at growing ends. Since one end of a treadmilling MT is shortening while the other is growing, CLIP-170 is expected to be present at the leading (plus) end but absent from the trailing (minus) end. This prediction was confirmed by direct dual label observation ( Fig. 5C). Thus, CLIP-170 can be used as a polarity marker for the growing MT plus end.
Polymer balance of treadmilling MTs comes at the level of individual MTs with shortening from the minus end being equal, on average, to growth at the plus end. However, in intact cells or centrosome-containing cytoplasts, minus ends are tethered at the centrosome. Since release from the centrosome and minus end shortening is infrequent ( Keating and Borisy, 1999; Waterman-Storer and Salmon, 1997; Vorobjev et al., 1999), most of the balance must come from the dynamics of the plus end. Therefore, the persistent growth of MT plus ends in the cell interior must be balanced primarily by shortening of other MT plus ends.
The steady state requirement of balance of growth and shortening holds true for every position in the cell, including the centrosome. Since nascent MTs are born at the centrosome at the rate of 5.6±2.3 per minute, we predicted that an equivalent rate of MT shortening back to the centrosome should be observed. To evaluate this issue, we randomly selected MTs having their plus ends close to the margin and monitored their fate ( Fig. 6A). In contrast to growth, shortening was not persistent. Shortening occurred at a velocity of 28.8±14.1 μm/minute (n=474; 10 cells) ( Fig. 6B,C). Frequency histograms of the distances shortened showed an approximately exponential decay ( Fig. 6D) with the mean distance being 2.9±3.4 μm. These properties are consistent with a first-order process of transition back to the growing state (rescue). The rescue frequency was determined to be 0.12 second-1 (91 transitions, 100 MTs, 735 seconds of observation) by tracking MTs shortening back from the margin until they began to grow. Thus, rescue as opposed to catastrophe was frequent leading to small shortening excursions being common and long ones rare. Nevertheless, shortening back to the centrosome could be directly observed, albeit at low frequency. Since MTs that shortened long distances were generally lost as their plus ends neared the congested area around the centrosome, we scored, as a more reliable estimate, the number of MTs that shortened by two-thirds of the cell radius. Out of all episodes of shortening, we obtained 6.4±2.7 per minute that shortened more than two-thirds of the distance to the centrosome.
Analysis of MT shortening. (A) Time-lapse sequence shows a rare event of plus end shortening (arrowheads) from the plasma membrane back to the centrosome within 12 seconds. (B) Histogram of instantaneous rates of MT shortening from the plasma membrane. (C) Length distribution of MT shortening episodes. MT ends adjacent to the cell margin were followed and the length of a continuous shortening episode was determined. The histogram shows small shortening episodes were frequent and long ones rare (n=319). (D) Time-lapse sequence shows release of a MT from the centrosome followed by shortening from the minus end (arrowheads). Time in seconds; bars, 5μ m.
Besides shortening from the plus end, infrequent releases of MTs from the centrosome also occurred. Released MTs had a short lifespan and rapidly depolymerized from the minus end ( Fig. 6E). In cytoplasts, the frequency of releases was 1.0±0.5 MT per centrosome per minute (58 releases, 11 cytoplasts). Thus, plus end death (6.4/minute) + minus end release (1.0/minute) was approximately equal to plus end birth (5.6 MTs/minute). This rough equality confirms that the MT system in CHO cells is indeed in steady state and it indicates that balance at the centrosome in CHO cells comes primarily (∼85%) from the plus end pathway with the minus end pathway making a minor (∼15%) but significant contribution.
The persistent growth of nascent MTs is an apparently paradoxical result because it is not clear how this behavior can be reconciled with the dynamic instability behavior observed near the cell margin. To address this question, we employed the conceptual framework of diffusion plus drift and the procedure of sequential subtraction analysis to obtain the necessary data.
Sequential subtraction images ( Fig. 7A) permit identification of growing and shortening MT ends as black or white line segments, respectively, even in regions of high MT density, which otherwise preclude direct observation of individual MTs. MTs that do not undergo a displacement (i.e. are stable or paused) between successive frames become canceled out and do not appear in the subtraction image.
Population analysis of MTs. (A) Sequential subtraction analysis for identification of growth and shortening. Image of Cy3-labeled MTs from a time-lapse series and corresponding differential image (In — In+1) obtained by subtracting from an image (In) the next image (In+1) in the series. Black and white segments represent MT growth and shortening, respectively, during the time interval. Five trapezoidal zones, each one-fifth of the cell radius, indicate the regions where growth and shortening events were scored. In the two right-hand panels, the fifth trapezoid is enlarged and growth and shortening excursions are shown in colors, — green and red, respectively. Parallel black and white segments arise from lateral shifts of MTs and were not scored. (B) Frequency distribution of growth and shortening velocities in region of 0.6-0.9 of cell radius fraction obtained by subtraction analysis. (C) Drift coefficient as calculated from histogram of growth and shortening velocities. Drift is positive and fairly uniform within the cell interior, dropping to negative values only near the cell boundary. (D) Distribution of MT ends. The position of active MT ends (growing or shortening) was scored by subtraction analysis and assigned to one of five trapezoidal zones shown in panel A (n=7421 episodes; 4244 growing; 3177 shortening; 4 cells). Data were fit to a single exponential function. The exponential functions taken separately for growth or shortening episodes were essentially identical.
or as the mean of the velocity histogram. Based on this histogram analysis, calculated values of the drift coefficient were ∼5 μm/minute in the cell interior, declining somewhat towards the cell margin ( Fig. 7C), indicating that throughout the cell interior, growth was strongly favored over shortening. Close to the cell margin (<3 μm) the calculated drift coefficient was negative (-2.7±0.4 μm/minute; n=235 growth events; 197 shortening events), reflecting the increase in proportion of shortening events because of the influence of the cell boundary. It should be noted that since paused MTs do not appear in the subtraction images, they do not contribute to the calculation of drift. As a check on the result, we also evaluated displacement histograms obtained from direct fluorescence imaging in the cell interior. Such histograms were also bimodal although now a population of paused MTs was detected. In the cell interior, paused MTs were infrequent (15%) while near the cell margin they were abundant (70%). In the cell interior, velocities and drift coefficients (data not shown) were similar to those obtained from subtraction analysis. However, near the cell margin, the larger fraction of pauses resulted in a proportionate decrease in the absolute value of the drift coefficient.
The diffusion coefficient represents fluctuations around the behavior predicted from drift and is calculated as the variance of the velocity frequency histogram. Calculation gave the apparent diffusion coefficient as 13.1±1.3 μm2/minute for the inner, 15.1±1.1μ m2/minute for the middle and 13.1±1.3μ m2/minute for the outer thirds of the cell, respectively. The ratio of coefficients, diffusion/drift was ∼3 μm and did not change significantly along the cell radius. As shown previously ( Vorobjev et al., 1999), positive drift predicts a non-random distribution of MT ends.
To determine the distribution of MT ends along the cell radius, the whole cell was divided into five radial zones. Counts of black or white line segments showed that MT ends, both growing and shortening, were rare near the centrosome and increased exponentially towards the cell margin ( Fig. 7D). Thus, in CHO cells, the length distribution of dynamic MT ends is an ascending one and most MTs are long. From an exponential curve fit to the data in Fig. 7D, the ratio of the coefficients was independently estimated as 5.8 μm, which is within a factor of 2 of the measured value. Thus, the diffusion plus drift framework is sufficient to account for the observed distribution of MT ends.
, we calculate drift coefficients of 4.4 μm/minute for the lamellae of NRK fibroblasts ( Mikhailov and Gundersen, 1998), 3.4 μm/minute for the periphery of CHO cells ( Shelden and Wadsworth, 1993), and 3.8 μm/minute for MTs growing parallel to the cell edge in newt lung cells ( Waterman-Storer and Salmon, 1997). Thus, an apparent imbalance of growth over shortening is a characteristic feature of many cultured cells. We conclude that persistent growth and positive drift are widespread features of MT dynamics.
In this study, we employed a combination of novel approaches to examine MT behavior in the cell interior. The results provide an updated picture of the MT life cycle in cells with a radial array originating from the centrosome ( Fig. 8A). A MT is born by nucleation on a centrosomal template, it grows through persistent elongation by addition of subunits at the plus end; in the vicinity of the cell margin, the MT undergoes fluctuations in length through episodes of plus end shortening and growing; and, finally, it dies when one of the stochastic episodes of shortening is of sufficient length to depolymerize it all the way back to the centrosome. An alternative path of death is release from the centrosome followed by persistent shortening from the minus end. In CHO and other fibroblasts, these properties combine to generate an apparent imbalance of growth over shortening (drift) for individual MTs in the cell interior that leads in the steady-state to a distribution of MT plus ends that steeply climbs as a function of position along the cell radius. This view of the MT life cycle and steady state differs from previous work in three ways: (1) that the default behavior of MTs in the cell interior is to grow persistently; (2) that MTs at the cell periphery show `dynamic instability' type behavior because of a boundary effect; and (3) that positive `drift' generates an ascending distribution of MT lengths.
MT life cycle and steady state length distribution (A). Life history of a typical MT reconstructed from data from two individual MTs. The first part (up to time axis break) traces persistent growth towards the cell boundary followed by catastrophes induced by the boundary that are rescued at high frequency. The second part (after time axis break) shows the rare event of shortening back to the centrosome. (B) Diagram of radial array of MTs showing distribution of lengths in accordance with exponential function fitted to data from subtraction analysis. Long MTs dominate over short ones.
The frequency of transition from growth to shortening of a MT in the cell interior was determined to be very low, 0.005 second-1, which equates to a half-time for the growing phase of 140 seconds. Assuming a radius of 20 μm for a typical CHO cell and a growth velocity for the MT plus end of 17 μm/minute, a MT would require only ∼70 seconds to grow the distance of the cell radius, which means that a nascent MT typically arrives at the cell margin before it has had a chance to shorten. Assuming a first-order process of transition to shortening, we calculate that an average of 72% of nascent MTs starting at the centrosome would still be in the growth phase by the time they reached the cell margin. This result is essentially the same as the experimentally observed value of 68% for cells. Cytoplasts are smaller than cells and therefore they have a higher percentage of MTs that arrive at the margin without shortening. Thus, persistent growth of a nascent MT can be understood as resulting from a combination of high elongation velocity and low catastrophe frequency. This remarkable feature of the MT life cycle was not previously recognized. The major explanation for this oversight is most likely that observations of MT dynamics in vivo have generally been made where it is technically easier to obtain the data — namely, at the cell periphery, where the cytoplasm is thin and MT density is relatively low, thus facilitating visualization of individual MTs. An implicit and untested assumption has been that MT dynamics parameters evaluated at the cell periphery are valid throughout the cell. Our results demonstrate that, at least in CHO and NRK cells, this is not the case.
Data consistent with persistent growth can be found in recent papers focusing on GFP-labeled plus end markers ( Perez et al., 1999; Mimori-Kiyosue et al., 2000a). Although these reports did not emphasize the persistent behavior of MT growth, they did contain data from which such conclusions could be drawn. In Vero cells, GFP-CLIP-170 tracks of 10 μm length were common ( Perez et al., 1999) and a similar picture emerges from movies of EB-1-GFP tracks in Xenopus A6 cells ( Mimori-Kiyosue et al., 2000a; supplementary material). Persistent growth as a property of MTs was also described in the fission yeast, S. pombe, where the majority of MTs grew until they reached the cell ends ( Brunner and Nurse, 2000). Taking these results together with our observations, we conclude that persistent growth of MTs in the cell interior is not an exceptional property of CHO cells. Rather, it may be an evolutionarily selected and widespread characteristic that has previously escaped notice because it is seen primarily in the cell interior.
As MTs neared the cell margin, their behavior changed to frequent alternation of brief phases of shortening and growth. Such behavior is how dynamic instability has come to be characterized in vivo. For example, growth and shortening lengths in PtK1 cells have been reported as 1.3 and 1.6 μm and in CHO cells as 3.2 and 4.3 μm, respectively ( Shelden and Wadsworth, 1993). We obtained similar values for CHO cells for MTs near the cell edge. Thus, the small duration of growth seen for MTs near the cell margin differed from the persistent growth in the cell interior.
What could be the explanation for this difference in behavior? Are MT dynamics intrinsically different in the cell interior as opposed to the cell margin or only apparently so? Closer inspection of MT behavior revealed that not all parameters depended on position in the cell. Velocities of growth and shortening (17 and 30 μm/minute, respectively) did not. We obtained the same values independently of whether the determinations were made at the cell margin or in the cell interior and they were similar to those obtained previously at the cell periphery ( Shelden and Wadsworth, 1993). Likewise, the rescue frequency was essentially constant. In the cell interior, the mean distance of shortening was small (2.9±3.4 μm) and the rescue frequency was high (0.12 second-1). Shortening of MTs back from the cell margin followed an exponential decay function, signifying that the probability of transition to the growing phase did not depend detectably upon the extent of shortening or position in the cell. The values of shortening length and rescue frequency were essentially the same as those obtained previously at the cell periphery (4.3±3.3 μm and 0.13 second-1, respectively) ( Shelden and Wadsworth, 1993). Finally, when we evaluated the apparent catastrophe frequency near the cell margin, we obtained a value (0.08 second-1), similar to that previously reported (0.06 second-1) (Sheldon and Wadsworth, 1993). Thus, the major difference in MT behavior is the apparent high frequency of catastrophe seen at the cell margin versus the low frequency (0.005 second-1) that we find in the cell interior.
We propose that the difference in catastrophe frequency is only apparent and can be accounted for by a `boundary effect' of the cell margin. The boundary prevents growth from continuing, inducing a pause and/or catastrophe. It follows that observations restricted to the cell periphery underestimate intrinsic growth duration and overestimate the catastrophe frequency. A high rescue frequency prevents MTs from shortening all the way back to the centrosome. Instead, shortening MTs are quickly converted to growing ones, which are then driven back to the cell membrane. Thus, the asymmetry of transition frequencies and the boundary effect combine to produce persistent growth in the cell interior but many small fluctuations of MT length near the cell margin.
What is the nature of the boundary? The simplest possibility is that the membrane itself or the dense actin network that typically underlies it presents a physical obstacle to growth. Alternatively, a chemical signal emitted from the cell surface could change the state of the MT. Growing MTs are marked by the presence of CLIP-170-family proteins (CLIP-170 and CLIP-115 for mammalian cells) ( Perez et al., 1999; Hoogenraad et al., 2000), CLASP ( Akhmanova et al., 2001), EB-1/APC complex ( Mimori-Kiyosue et al., 2000a; Mimori-Kiyosue et al., 2000b) and dynactin complex ( Vaughan et al., 1999) at their plus ends. The relationship between the presence of these proteins and MT dynamics has yet to be established for mammalian cells. CLIP-170 family proteins could be good candidates for ensuring MT growth in the cell interior, as the fission yeast ortholog of CLIP-170, tip1p, stabilizes MT growth by preventing premature catastrophe ( Brunner and Nurse, 2000). EB1/APC complex, which promotes MT polymerization in vitro ( Nakamura et al., 2001), and increases MT stability in vitro and in vivo ( Zumbrunn et al., 2001), might also prevent growing MT tips from depolymerization. If any or all of these proteins were required to maintain a low catastrophe frequency or a high rescue frequency and if a factor at the cell surface caused them to dissociate from the MT plus end, the result would be an induced transition to the shortening phase.
The simplest criterion of steady state is that the number and amount of MT polymer does not change over time. An apparently paradoxical result is that in the steady state some MTs persistently grow. Where does the shortening come from to balance the growth? Fig. 8A provides a qualitative explanation. Neglecting the minor contribution that the minus end pathway plays in CHO cells, the growth of a nascent MT is essentially compensated by the complete shortening of another MT from the plus end. Fluctuations of growth and shortening near the cell margin, on average, equate to each other because persistent growth up to the cell boundary restores polymer lost by shortening from the boundary. Thus, cellular levels of MT polymer remain constant in the presence of persistent growth of individual MTs.
A deeper paradox is the ascending distribution of MT ends. Why is the distribution not uniform in the cell? The answer is again related to a boundary effect. Persistent growth and non-persistent shortening of MTs is equivalent to stating that the catastrophe frequency is small and the rescue frequency is high. In CHO cells, the catastrophe frequency is so small that nascent MTs typically arrive at the cell margin before having had a chance to shorten. In contrast, MTs typically shorten for only a small distance before being driven back to the cell margin. Since, by definition, a boundary imposes a limit to growth, the asymmetry in transition frequencies will cause MT ends to spend more time and thus to accumulate near the cell margin ( Fig. 8B).
, where n refers to the number of MT ends at any given plane in the cell and∂ n/∂x is the concentration gradient of ends across the plane.
. This signifies that there will be 20 times more MT ends within 5 μm of the cell margin than within a 5 μm zone around the centrosome.
The asymmetry of MT dynamics in vivo stands in contrast to MTs in vitro, where both growth and shortening tend to be persistent ( Mitchison and Kirschner, 1984b; Horio and Hotani, 1986; Walker et al., 1988). The difference between parameters observed in vitro and in vivo suggest that there is a regulatory mechanism at the MT plus end. Several proteins have been suggested to increase the catastrophe frequency including XKCM1 ( Walczak et al., 1996; Desai et al., 1999; Tournebize et al., 2000) and stathmin/Op18 ( Belmont and Mitchison, 1996; Howell et al., 1999). Some MT-associated proteins (MAP4, Xenopus XMAP 215 and its human homolog TOGp) can play an opposite role, rescuing MTs by stabilization and/or assembly-promotion ( Tournebize et al., 2000; Spittle et al., 2000; Popov et al., 2001) (for reviews, see Andersen, 2000; Heald, 2000). In addition, plus end binding proteins such as CLIP-170 ( Perez et al., 1999) or EB1 ( Mimori-Kiyosue et al., 2000b) represent potential regulatory factors, although mechanistic studies demonstrating how they might affect MT dynamics are lacking.
Growth of nascent MTs in CHO cells was not only persistent but rapid. The velocity we obtained, 15-20 μm/minute, was higher than most values reported for MT growth in other cells, cell extracts or for purified tubulin (reviewed by Odde and Buettner, 1995). However, similar instantaneous values were reported for CHO cells ( Shelden and Wadsworth, 1993), GFP-CLIP-170 tracks in Vero cells ( Perez et al., 1999) and EB-1-GFP tracks in Xenopus A6 cells ( Mimori-Kiyosue et al., 2000a). It is not clear what factors are responsible for the differences in reported rates but our study and the several reports cited indicate that MT growth in vivo can be astonishingly rapid. The rates in vivo are almost an order of magnitude higher than those predicted by in vitro studies of pure tubulin ( Walker et al., 1988). This consideration suggests that the rate constant for MT growth in vivo is higher than that determined in vitro or that the cell has a mechanism to maintain the free tubulin pool substantially above the critical concentration.
The life cycle of MTs in CHO and NRK cells displays several characteristic features including persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary. What might be the biological significance of this set of properties? We suggest that the answer is to provide a mechanism by which the MT array can rapidly `sense' changes of the cell periphery. Rapid and persistent growth allows nascent MTs to elongate from the centrosome to the cell boundary in a short time. This would be advantageous for a locomoting cell in which the cell margin is advancing in that the MT cytoskeleton could rapidly accommodate to any protrusion or change in cell shape. Such accommodation could also be important for maintenance of membrane trafficking within the cell, including transport of vesicles either to or away from the cell surface. Finally, efficient sensing of the periphery by MTs may be important for coordinating motile activities around the cell perimeter to achieve directional motility.
The new insights into MT dynamics obtained from this analysis of the MT life cycle in CHO and NRK cells suggest that similar comprehensive analyses in other cell types will be rewarding. It will be important to determine whether different cell types such as fibroblasts, epithelial or neuronal cells have similar or distinctive MT `lifestyles'. In any event, this study points out the critical necessity of carrying out determinations of MT behavior in the cell interior and in recognizing that the cell is a finite system in which its boundary exerts important effects.
We thank I. Maly, T. Svitkina, E. Taylor and G. Albrecht-Buehler for helpful discussions and critical reading of the manuscript, V. Rodionov for help with double-label experiments. This work was supported by NIH Grant GM 25062 to G.G.B., Fogarty International Research Collaboration Award TW-00748 and U.S. Civilian Research and Development Foundation Award RB1-2025 to G.G.B. and I.A.V.
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TLJ News: October 26-31, 2012.
10/31. The Federal Communications Commission (FCC) published a notice in the Federal Register (FR) that sets comment deadlines for its Further Notice of Proposed Rulemaking (NPRM) [146 pages in PDF] regarding its program access rules. The FCC adopted and released this item on October 5, 2012. It is FCC 12-123 in MB Docket No. 12-68. The deadline to submit initial comments is November 30, 2012. The deadline to submit reply comments is December 17, 2012. See, FR, Vol. 77, No. 211, October 31, 2012, at Pages 66052-66065. The FCC also published a notice in the FR that announces, describes, and sets the effective date for its Report and Order (R&O). The effective date is November 30, 2012. See, FR, Vol. 77, No. 211, October 31, 2012, at Pages 66025-66051. See also, stories titled "FCC Lets Expire Its Per Se Ban on Exclusive Program Distribution Contracts", "FCC Adopts Report and Order on Program Access Rules", "FCC Adopts NPRM on Case by Case Analysis of Exclusive Contracts", and "Reaction to FCC's Program Access Order" in TLJ Daily E-Mail Alert No. 2,460, October 6, 2012.
10/31. The National Institute of Standards and Technology's (NIST) Computer Security Division (CSD) released its draft SP 800-164 [33 pages in PDF] titled "Guidelines on Hardware-Rooted Security in Mobile Devices". The deadline to submit comments is December 14, 2012.
10/29. The Supreme Court heard oral argument in Clapper v. Amnesty International, Sup. Ct. No. 11-1025. At issue is whether the plaintiffs have standing to challenge the constitutionality of the "outside the United States" surveillance provision of the Foreign Intelligence Surveillance Act (FISA).
Introduction. Jameel Jaffer of the American Civil Liberties Union (ACLU) argued for Amnesty International (AI) and the other plaintiffs. Donald Verrilli, the Solicitor General, argued for the government surveillance agencies.
Verrilli said that the statute at issue provides "authority to the executive to conduct surveillance targeted at foreign persons located abroad for foreign intelligence purposes". However, the Justices focused on persons within the US, particularly attorneys representing clients abroad, who are also affected by this surveillance power.
Justice Elena Kagan said that the "statute greatly expands the government's surveillance power". Justice Anthony Kennedy said that it provides an "extraordinarily wide-reaching power".
However, while the complaint filed in the District Court challenges the constitutionality of the 2008 statute, the issue now before the Supreme Court is merely whether the plaintiffs have standing. Since the government has not notified the plaintiffs that that their phone conversations have been intercepted or emails copied, the government argues that they have not shown that they have suffered an injury that would confer standing to sue.
The question presented is "Whether respondents lack Article III standing to seek prospective relief because they proffered no evidence that the United States would imminently acquire their international communications using Section 1881a-authorized surveillance and did not show that an injunction prohibiting Section 1881a authorized surveillance would likely redress their purported injuries."
10/26. President Obama signed an executive order (EO) titled "Establishing the White House Homeland Security Partnership Council". The most notably characteristic of this EO is that it does not disclose what it is intended to accomplish.
This EO creates a "Council" and a "Steering Committee" related to "homeland security", but is otherwise carefully worded to obfuscate its purpose.
This order provides that at a later date the Steering Committee, which will be comprised solely of representatives of federal executive branch departments, will "the determine the scope of issue areas the Council will address".
President Obama has suggested that he might issue an executive order pertaining to cyber security that would attempt to impose the regulatory regime described in S 3414 [LOC | WW], the "Cybersecurity Act of 2012". That bill has stalled in the Senate, and been ignored by the House Republican leadership. See, story titled "Senate Rejects Cloture on Sen. Lieberman's Cyber Security Bill" in TLJ Daily E-Mail Alert No. 2,419, August 3, 2012.
See also, stories titled "House and Senate Republicans Write Obama Opposing Regulation of Internet by Executive Order" and "Sen. Grassley States Cyber Security Should Be Addressed by Legislation Rather Than Presidential Order" in TLJ Daily E-Mail Alert No. 2,461, October 15, 2012.
However, while this EO references "homeland security", it makes no reference to "cyber security", "internet", "computer systems", or related things.
This EO does reference a "National Security Strategy". The Executive Office of the President (EOP) released a document [60 pages in PDF] in May of 2010 with this title. Moreover, this EO contains a quotation taken from page 16 of that document. That 2010 document addresses many subjects, including cyber security.
This EO may be intended as an implementation of one aspect of that 2010 statement.
This EO may be an insignificant effort to present the mere appearance of greater administration activity.
This EO may be an effort to evade laws that are specific to individual executive departments, by transferring activity to an extra-departmental body. It might likewise be an effort to evade the committee based Congressional oversight system.
This EO may be part of an effort to attain policy objectives via executive order, and extra-departmental processes, that the administration has not been able to implement via legislation, for example, in the areas of cyber security or data retention, as well as in other homeland security policy areas that are not technology related.
This EO is also published in a notice in the Federal Register, Vol. 77, No. 213, November 2, 2012, at Pages 66351-66355.
10/26. The Librarian of Congress released a notice in the Federal Register (FR) that adopts the fifth set of triennial rules containing exemptions to the anti-circumvention provisions § 1201.
The Library of Congress's (LOC) Copyright Office (CO) released its report [169 pages in PDF] containing proposed rules on October 12, 2012. The final rule follows the proposals contained in this report.
an exemption for circumventing literary works distributed electronically, to permit blind and other persons with print disabilities to use screen readers and other assistive technologies.
an exemption to allow jailbreaking mobile phones (but not tablets) -- that is, an exemption for circumventing computer programs on wireless telephone handsets, to enable interoperability of software applications.
an exemption to allow unlocking mobile phones -- that is, exemption for circumventing computer programs on wireless phones that were acquired within ninety days of the effective date of the exemption, for the purpose of connecting to alternative networks.
two related exemptions allowing the circumvention of movies on DVDs and delivered via online services, to permit the use of short portions for purposes of certain noncommercial criticism and comment.
an exemption for circumventing movies on DVDs or distributed by online services, for the purpose of research to create players capable of rendering captions and descriptive audio for persons who are blind, visually impaired, deaf or hard of hearing.
The CO and Librarian of Congress also rejected numerous proposed exemptions.
For example, the CO and Librarian rejected a proposed exemption for "Literary works in the public domain that are made available in digital copies" advocated by the Open Book Alliance (OBA). This would have impacted Google's book digitization program. Although, the opposition came from copyright sector groups that argued that such works also often contain ancillary copyrightable elements.
The Public Knowledge (PK), a constant critic of § 1201, expressed its dismay that the CO and Librarian rejected its proposal for an exemption for circumvention of DVDs for space shifting.
These new rules take effect on October 28. The triennial rules are codified at 37 C.F.R. § 201.40.
certain movies on DVD protected by CSC.
certain programs that enable wireless phones to execute software applications.
programs that enable used wireless phones to connect to a wireless telecommunications network if allowed by the network operator.
certain video games, for the purpose of investigating or correcting security flaws.
certain programs protected by dongles.
e-book when all e-book editions of the work do not allow read aloud functions.
See, FR, Vol. 77, No. 208, October 26, 2012, at Pages 65260-65279.
Anti-Circumvention and Triennial Exemptions. The anti-circumvention provisions of the DMCA are codified at 17 U.S.C. § 1201, et seq. Subsection 1201(a)(1)(A) provides that "No person shall circumvent a technological measure that effectively controls access to a work protected under this title ...".
Then, § 1201(a)(2)(A) provides that "No person shall manufacture, import, offer to the public, provide, or otherwise traffic in any technology, product, service, device, component, or part thereof, that --- (A) is primarily designed or produced for the purpose of circumventing a technological measure that effectively controls access to a work protected under this title;"
Furthermore, § 1201(b)(1)(A) provides that "No person shall manufacture, import, offer to the public, provide, or otherwise traffic in any technology, product, service, device, component, or part thereof, that --- (A) is primarily designed or produced for the purpose of circumventing protection afforded by a technological measure that effectively protects a right of a copyright owner under this title in a work or a portion thereof; ..."
Then, Subsections 1201(a)(1)(B) through (E) provide for rulemaking proceedings conducted by the CO every three years to establish exemptions to the prohibition of (a)(1)(A) for certain non-infringing uses.
History of This Proceeding. On September 29, 2011, the CO published a Notice of Inquiry (NOI) that initiated this fifth triennial proceeding, and requested proposals for exemptions. See, notice in the Federal Register, Vol. 76, No. 189, September 29, 2011, at Pages 60398-60405. See also, story titled "Copyright Office Opens 5th Triennial Proceeding on DMCA Anti-Circumvention Exemptions" in TLJ Daily E-Mail Alert No. 2,306, September 29, 2012.
On December 20, 2011, the CO published a Notice of Proposed Rulemaking (NPRM). See, notice in the Federal Register, Vol. 76, No. 244, December 20, 2011, at Pages 78866-78868. See also, stories titled "Copyright Office Seeks Comments on Proposed Anticircumvention Exemptions" and "Summary of Proposed Exemptions to Anticircumvention Provisions § 1201" in TLJ Daily E-Mail Alert No. 2,329, January 16, 2012.
The CO then held a series of public hearings in Washington DC and Los Angeles, California, in May and June of 2012.
The CO released its recommendations for the Librarian of Congress on October 12.
The just released rules are the fifth set of triennial exemptions adopted since enactment of the DMCA in 1998. These rules take effect on October 28, 2012.
The Exemptions. The just released notice states that "Upon the recommendation of the Register of Copyrights, the Librarian has determined that the following classes of works shall be exempt from the prohibition against circumvention of technological measures set forth in Section 1201(a)(1)(A):"
First: "Literary works, distributed electronically, that are protected by technological measures which either prevent the enabling of read-aloud functionality or interfere with screen readers or other applications or assistive technologies, (i) when a copy of such a work is lawfully obtained by a blind or other person with a disability, as such a person is defined in 17 U.S.C. 121; provided, however, the rights owner is remunerated, as appropriate, for the price of the mainstream copy of the work as made available to the general public through customary channels; or (ii) when such work is a nondramatic literary work, lawfully obtained and used by an authorized entity pursuant to 17 U.S.C. 121."
Second: "Computer programs that enable wireless telephone handsets to execute lawfully obtained software applications, where circumvention is accomplished for the sole purpose of enabling interoperability of such applications with computer programs on the telephone handset."
Third: "Computer programs, in the form of firmware or software, that enable a wireless telephone handset originally acquired from the operator of a wireless telecommunications network or retailer no later than ninety days after the effective date of this exemption to connect to a different wireless telecommunications network, if the operator of the wireless communications network to which the handset is locked has failed to unlock it within a reasonable period of time following a request by the owner of the wireless telephone handset, and when circumvention is initiated by the owner, an individual consumer, who is also the owner of the copy of the computer program in such wireless telephone handset, solely in order to connect to a different wireless telecommunications network, and such access to the network is authorized by the operator of the network."
Fourth and fifth: a pair of lengthy and related exemptions for motion pictures on DVDs or distributed by online services, for purposes of criticism or comment in noncommercial videos, documentary films, nonfiction multimedia ebooks offering film analysis, and for certain educational uses by college and university faculty and students and kindergarten through twelfth grade educators.
Sixth: "Motion pictures and other audiovisual works on DVDs that are protected by the Content Scrambling System, or that are distributed by an online service and protected by technological measures that control access to such works, when circumvention is accomplished solely to access the playhead and/or related time code information embedded in copies of such works and solely for the purpose of conducting research and development for the purpose of creating players capable of rendering visual representations of the audible portions of such works and/or audible representations or descriptions of the visual portions of such works to enable an individual who is blind, visually impaired, deaf, or hard of hearing, and who has lawfully obtained a copy of such a work, to perceive the work; provided however, that the resulting player does not require circumvention of technological measures to operate."
10/26. The Department of Homeland Security (DHS) announced in a release that the U.S. and Canada have a "Cybersecurity Action Plan". The DHS did not release the text of any plan. The DHS release states that this plan "outlines shared goals for improved engagement, collaboration, and information sharing at the operational and strategic levels, with the private sector, and in public awareness activities."
Go to News from October 21-25, 2012.


§ 126-3 Annual dumpster permit required.
§ 126-4 Application for permit; fee.
§ 126-5 Location, use and conditional requirements.
§ 126-6 Revocation of permit.
§ 126-7 Failure to abate violations.
§ 126-8 Penalties for offenses.
§ 126-9 Code Enforcement Officer to serve as enforcement agent.
Littering — See Ch. 205.
Nuisances — See Ch. 221.
Solid waste — See Ch. 283.
Storage containers — See Ch. 177.
This chapter is adopted for the protection and promotion of the public health, safety and general welfare of the community by regulating the use, maintenance and operation of bulk garbage containers referred to as "dumpsters."
A bulk storage container for garbage and other waste materials that can be hauled directly to the point of disposal or emptied into a large compactor-type truck or other conveyance for disposal.
No person, partnership, corporation or other entity shall own, lease or arrange for the emplacement or use of a dumpster within the Village of Lancaster, unless a permit therefor shall be secured from the Village of Lancaster. Persons, partnerships, corporations or other entities which arranged for the emplacement or utilization of a dumpster within the Village of Lancaster as of the effective date of this chapter shall apply for a permit within 60 days of the effective date of this chapter. Although in accordance with § 126-4A(5), a permit may be issued for a lesser duration, no permit shall be granted for a period exceeding one year.
The name and address and phone number of the applicant and of the owner or owners of the parcel upon which the dumpster shall be situate and, if applicable, the lesser of the dumpster.
A description or diagram of the locale of the dumpster.
A description of the dumpster, which shall include its dimensions and capacity.
The circumstances necessitating the use of the dumpster.
The length of time the applicant intends to utilize the dumpster.
The frequency with which the dumpster will be emptied.
That the applicant has taken notice of and consents to the application of § 126-7 of this chapter.
All applications for permits shall be accompanied by a fee as set forth at Chapter 144 of the Code of the Village of Lancaster, New York.
No dumpster shall be located in or on a public right-of-way.
Garbage and other waste materials must be completely contained within the dumpster; no accumulation of garbage or waste materials will be permitted outside the confines of the dumpster.
Dumpsters shall be kept in good repair, be structurally sound, be leakproof and be easily opened and closed. Dumpsters shall be painted as required to prevent the show of rust and deterioration.
Dumpsters shall be positioned and constructed so as to stand firmly upright.
The name and phone number of the holder of the permit issued pursuant to § 126-3 of this chapter shall be printed clearly and legibly on each dumpster.
Open-top dumpsters shall be enclosed with a fence composed of wood lattice, fine mesh fencing or other suitable materials as determined by the Village of Lancaster, with a minimum height of three feet above the top of the container. In the case of open-top dumpsters, use of which is limited to the collection of waste materials associated with activities described in § 104-8A of the Code of the Village of Lancaster, New York, the Village Board of the Village of Lancaster shall retain discretion to waive the requirements set forth within this subsection.
The holder of the permit issued pursuant to § 126-3 of this chapter shall arrange to have the dumpster emptied on such a schedule so that garbage and other waste materials do not accumulate so as to overflow the confines of the dumpster, restrict the closing of the dumpster cover or cause offensive odors or otherwise create a health hazard.
Dumpsters with lids shall be kept closed and secured at all times except when necessary to deposit or remove debris.
The location and use of a dumpster shall conform to all state and local fire codes.
Any permit issued pursuant to § 126-3 of this chapter may be summarily revoked by the Village of Lancaster if the subject dumpster is placed or utilized in a manner contrary to information included within the permit application or in violation of any provision or § 126-5 of this chapter.
If the dumpster is utilized in violation of any of the provisions of § 126-5 of this chapter, the Village may enter the property to remove, eliminate, repair or otherwise abate the violation. For this purpose the Village may use its own personnel or may hire a contractor. The cost incurred whether Village personnel are used or a contractor is hired will be charged to the permit holder. If a permit is revoked pursuant to the provisions of § 126-6, reissuance of a permit may be conditioned upon payment by the applicant of the cost incurred by the Village of Lancaster pursuant to this section.
In addition to any other remedy provided by this chapter, any person committing an offense against any provision of this chapter shall, upon conviction thereof, be punishable as provided in Chapter 1, General Provisions, Article II, Penalties for Offenses, of the Code of the Village of Lancaster.
The Code Enforcement Officer shall be empowered to act as the agent of the Village of Lancaster in administrating and enforcing compliance with this chapter.


As I have previously noted, I sympathize with the difficulties faced by MassDEP in trying to implement the SJC decision in Kain. However, that does not mean that MassDEP can simply take the easy way out. After rereading Kain, I have come to the conclusion that DEP’s proposal to limit GHG emissions from electric generating facilities in Massachusetts would in fact violate Kain, rather than constitute a means of compliance with Kain.
MassDEP argued, in part, that the RGGI regulations, promulgated under § 3(c) of the GWSA, satisfied the 3(d) requirement. The SJC disagreed, but the basis for its disagreement is important and I don’t think that MassDEP has read Kain as carefully as it should have. In short, I think that the SJC concluded that §§ 3(c) and (d) are separate and distinct. In other words, while regulations under § 3(c) do not satisfy § 3(d), it is also true that the two sections are intended to regulate different types of sources, and that electric generating sources are simply not subject to regulation under § 3(d).
[The GWSA], § 3 (c), specifically carves out a separate process by which emissions levels and limits associated with the electric sector are established in consultation with the secretary and the Department of Energy Resources and are to take into account the RGGI. By doing so, the Legislature recognized that a significant part of the electric sector would already be subject to regulations associated with the RGGI. The RGGI is also addressed extensively in G. L. c. 21A, § 22, lending further support to the conclusion that the Legislature intended to treat emission reductions associated with the electric sector differently from other reductions in other sectors of the economy.
I’m sorry, but this sure sounds to me as though the SJC understands the GWSA to regulate electric generating sources under § 3(c) and all other sources under § 3(d). Here’s a rhetorical question that makes the point. How could the electric sector be treated differently from regulations promulgated under § 3(d) if in fact it is regulated under § 3(d)?
Finally, this reading has the virtue of making the SJC decision Kain less at odds with the actual goals of the GWSA. As the Court noted, the legislature acknowledged RGGI in § 3(c). Why would the legislature “specifically carve out a separate process by which emissions levels and limits associated with the electric sector are established” if it anticipated that the electric sector could also be regulated under § 3(d) in a way that is fundamentally inconsistent?
This is a case I’d enjoy litigating.


In many cases, a client may suggest the attorney do something. Sometimes, the suggestion is very good and the attorney follows the suggestion. Other times, the client tells the attorney to do something and the attorney, because of his or her experience and training, tells the client no. The attorney then may explain why such a suggestion is not a good idea, based on his experience, knowledge of the case facts, knowledge of the judge on the case, the prosecutor assigned, criminal procedure and how the laws of evidence apply to the charges at issue.
This article is written to prevent a client from hurting himself or herself based on such a belief that the role of an attorney is to follow the commands of the client. It is not meant to defend the attorney’s decisions, as the client actually may have a very helpful suggestion after all, but clarify the authority that the attorney has in handling the case and specify those areas that the client retains absolute authority over in the defense of a criminal case. California Rules of Professional Conduct, Rule 2-100.
Counsel for a criminal defendant, generally speaking, controls all decisions regarding trial tactics and court proceedings. People v. Masterson (1994) 8 Cal.4th 965, 35 Cal.Rptr. 2d 679; People v. Whittington (1977) 74 Cal.App.3d 806, 820, 141 Cal.Rptr. 742. What exactly are “trial tactics?” Obviously, this would include things such as whether to give an opening statement (or not), when to exercise peremptory challenges during voir dire and what experts to retain for trial.
5. Whether to waive time to trial within 60 days of the superior court arraignment (Penal Code § 1382(a)(2)). However, there are limitations. For example, the waiver cannot be simply to accommodate defense counsel’s calendar if it results in trial beyond statutory deadlines (People v. Johnson (1980) 26 Cal.3d 557, 568, 162 Cal.Rptr. 431).
7. Whether to request a change of venue (People v. Guzman (1988) 45 Cal.3d 915, 937, 248 Cal.Rptr. 467, overruled on other grounds in Price v. Superior Court (2001) 25 Cal.4th 1046, 1069, n 13, 108 Cal.Rptr.2d 409).
4. Whether to demand a jury trial (People v. Holmes (1960) 54 Cal.2d 442, 5 Cal.Rptr. 81).
Why Hire a Private Attorney? Why Shouldn't One Use the Public Defender?


A "wager" has been defined as a contract conditional upon an event in which the parties have no interest except that which they create by the wager;3 but this attempts to limit the term to contracts not permitted by law, and is not broad enough. Parties may make a wager on a matter in which they are interested. It is more accurate to say that a wager is a promise to pay money or transfer property upon the determination or ascertainment of an uncertain event or fact, the consideration for the promise being either a present payment or transfer by the other party, or a promise to do so upon the event or fact being determined or ascertained in a particular way.4 The term is often applied to contracts not permitted by law, as opposed to others which, though precisely similar in their nature, may be enforced, and this has resulted in some confusion.
2Farmers' & Mechanics' Nat. Bank v. Dearing, 91 U. S. 29, 23 L. Ed. 196. Bee "Banks and Banking" Dec. Dig. (Key-No.) § 270; Cent. Dig. §§ 1023-105S.
3Leake, Cont 377. By the terras of a note, given In part payment of land, defendant promised to pay .$900 if cotton should rise to 8 cents by a certain date, and, if not, to pay $500. It was held that the agreement was not a wager on the price of cotton, "for the parties had an interest in the contingency. The defendant purchased the land at the lowest price, unconditionally, but contracted to pay a larger sum if the value should be enhanced by the increased value of its product" Ferguson v. Coleman, 3 Rich. (S. C.) 99, 45 Am. Dec. 761. A contract by which a party purchases 50 bushels of "Bohemian oats" at $10 a bushel, and the seller agrees to sell for him the next year 100 bushels at $10 a bushel, has been held not to be a gambling contract. Shipley v. Reasoner, 80 Iowa, 548, 45 N. W. 1077; Matson v. Blossom, 50 Hun, 000, 2 N. Y. Supp. 551: Contra, Schmueckle v. Waters, 125 Ind. 205, 25 N. E. 2S1. Such contracts, however, are illegal, since they cannot be performed without defrauding some one. Hanks v. Brown, 79 Iowa, 560, 44 X. W. 811; Merrill v. Packer. 80 Iowa, 542, 45 N. W. 1076. Contra, Matson v. Blossom, supra. For similar contract, illegal because tending to defraud, see Hubbard v. Freiburger, 133 Mich. 139, 94 N. W. 727. See •'Gaming," Dec. Dig. (Key-No.) § 1; Cent. Dig. § I.
4 Anson, Cont. (4th Ed.) 173; Hampden v. Walsh, 1 Q. B. Div. 189. See "Gaming," Dec. Dig. (Key-No.) § 1; Cent. Dig. § 1.
5 Anson, Cont (4th Ed.) 174-176.
6 See Gilbert v. Sykes, 16 East, 150; Hartley v. Rice, 10 East, 22; Good v. Elliott, 3 Term R. 693; Eltbam v. Kingsman, 1 Barn. & Ald. 683; Atherford v. Beard, 2 Term R. 610; Evans v. Jones, 5 Mees. & W. 77; Ditchburn v. Goldsmith, 4 Camp. 152. And see Brogden v. Marriott, 3 Bing. N. C. 88; Ramloll v. Soojumnull, 6 Moore, P. C. 310; Bunn v. Riker, 4 Johns. (N. Y.) 426, 4 Am. Dec. 292; Rust v. Gott, 9 Cow. (N. Y.) 169, 18 Am. Dec. 497; Hill v. Kidd, 43 Cal. 615; Vischer v. Yates, 11 Johns. (N. Y.) 23. See "Gaming," Dec. Dig. (Key-No.) §§ 1-5; Cent. Dig. §§ 1-15.
7 Campbell v. Richardson, 10 Johns. (N. Y.) 406; Good v. Elliott, 3 Term R. 693; Clendining v. Church, 3 Caines (N. Y.) 141; Winchester v. Nutter, 52 N. H. 507, 13 Am. Rep. 93; Dewees v. Miller, 5 Har. (Del.) 347; Stoddard v. Martin, 1 R. I. 1, 19 Am. Dec. 643; Buchanan v. Insurance Co., 6 Cow. (N. Y.) 318; Wheeler v. Spencer, 15 Conn. 28; Johnston v. Russell, 37 Cal. 670; Wroth v. Johnson, 4 Har. & McH. (Md.) 284; Cothran v. Ellis, 125 I11. 496, 16 N. E. 646; Trenton Mut. Life & Fire Ins. Co. v. Johnson, 24 N. J. Law, 576; Wootan v. Hasket, 1 Nott & McC. (S. C.) 180; Kirkland v. Randon, 8 Tex. 10, 58 Am. Dec. 94. Wager as to shape of earth, Hampden v. Walsh, 1 Q. B. Div. 189; as to weight of hog, Mulford v. Bowen, 9 N. J. Law, 315; as to result of past election in another state, Smith" v. Smith, 21 I11. 244, 74 Am. Dec. 100; as to time within which railroad would be completed, Beadles v. Bless, 27 I11. 320, 81 Am. Dec. 231; Johnson v. Fall, 6 Cal. 359, 65 Am. Dec. 518. Bee "Gaming," Dec. Dig. (Key-No.) §§ 1-5; Cent. Dig. §§ 1-15.
There are now, both in England 9 and in this country, statutes covering the subject. There is so much difference in the statutes of the different states that it would be impracticable to attempt to give them. It is sufficient to say that in almost all the states, if not in all, the statutes make all gambling contracts either void, or both illegal and void.