PCs undergo independent processive motions during early meiotic prophase
At the onset of meiotic prophase, all PCs associate with the NE, where they induce the aggregation of the transmembrane proteins SUN-1 and ZYG-12 into patches ranging from 0.3 to 1.2 µm in diameter (; Penkner et al., 2007
; Sato et al., 2009
; Baudrimont et al., 2010
; Harper et al., 2011
). These patches persist until the completion of synapsis, at which point SUN-1 and ZYG-12 redistribute throughout the NE.
To analyze the motion of PCs, we first recorded images from animals expressing a zyg-12::gfp
transgene (Malone et al., 2003
). Initial observations revealed that the ZYG-12::GFP patches observed in early meiotic prophase were highly mobile (Sato et al., 2009
). Using the OMX (Optical Microscope Experimental) high-speed wide-field imaging system (Carlton et al., 2010
), we recorded single-wavelength 3D data stacks, with 2-s intervals between successive stacks (; and Video 1
). These recordings were limited to a duration of ~5 min before the signal/noise ratio was compromised by photobleaching. The resulting 4D datasets allowed segmentation and tracking of fluorescent patches along the nuclear surface using a semiautomated approach (see Materials and methods).
These recordings provided several insights into the motion of PCs during early prophase. First, the number of patches observed typically ranged from four to six per nucleus, fewer than the 12 individual chromosomes. This is consistent with evidence that homologous PCs pair early in prophase, based on immunofluorescence and FISH analysis (MacQueen et al., 2002
; Phillips et al., 2005
). The detection of fewer patches than chromosome pairs also suggests that chromosomes interact with both homologous and heterologous partners from the earliest stages at which patches are observed. Patches were frequently observed to merge and/or split over the course of a few minutes. An example of a nucleus in which two patches merge, remain together for 6 s, and subsequently separate can be seen in and Video 2
. An example of a nucleus in which six patches are clearly moving independently is shown in and Video 2. Quantitative analysis of patch trajectories indicated that the motions were largely uncorrelated in their xyz direction and are therefore not a consequence of nuclear translation or rotation (Fig. S1
). Although we observed many instances of multiple ZYG-12 patches in proximity, these clusters were transient and were not preferentially localized to one area of the NE, corroborating previous analysis of fixed samples, which indicated that attachment sites do not form a classical tightly clustered bouquet ().
The distribution of ZYG-12 step sizes was extremely heterogeneous, both within the population and for individual trajectories (). The mean xyz step size for all patches between adjacent data stacks acquired at 2-s intervals was 0.25 µm, corresponding to a mean apparent speed of 0.125 µm/s. However, we observed occasional jumps of >0.8 µm within a 2-s interval, indicating the occurrence of transient movements of ≥0.4 µm/s. At this 2-s sampling interval, we detected only a few instances in which the direction and rate of motion of individual patches were correlated over consecutive time points (Fig. S1). This weak correlation suggested that changes in direction and speed occur on a time scale more rapid than our sampling rate and that data collected at higher temporal resolution would enable a more complete description of patch motion.
Figure 2. Rapid 2D imaging enables two modes of motion to be distinguished. (A) Plots depicting xyz step sizes between each time point for three patches shown in . Plot colors correspond to patch color in time-lapse images. (B) Trajectory of a single (more ...)
We acquired data at a fivefold higher sampling rate by recording a single confocal optical section at 400-ms intervals, capturing multiple patches per nucleus (Video 3
). In these 2D datasets, we detected a marked increase in directional correlation between adjacent patch movements (Fig. S1), indicating the occurrence of processive movements of a few seconds duration. This revealed that patch trajectories are composites of at least two distinct modes of displacement: PCMs, in which patches move continuously in the same direction for up to several seconds, interspersed with periods in which patches exhibit constant changes in direction and remain close to their origins (, bold segments denote PCMs). Although observed step sizes in our data did not change markedly during a PCM (, blue), these two modes of motion could be clearly distinguished by tracking the distance traveled by a patch from its initial position over time (, red plot): periods in which the displacement shows uniform and concerted changes over several time points stand out among the small fluctuations in the otherwise horizontal plot. To automatically identify these PCMs, we searched for segments in which the cumulative displacement over three consecutive time intervals (1.2 s) was in the top 15th percentile of three-step displacements ( [bold regions] and C [shaded regions]). Modifying these criteria by including a range of two to five consecutive steps or a displacement percentile range of 5–25% did not markedly affect our results (unpublished data). Using these criteria, we identified 91 individual PCMs from 36 trajectories in five datasets.
The measured velocities during PCMs conformed to a tight normal distribution, with a mean speed of 0.19 µm/s (SD = 0.07 µm/s; ). The distributions of the duration of PCMs and total distance traveled during each instance exhibited exponential decay, characteristic of processive, motor-driven motions measured both in vivo and in vitro (e.g., Vale et al., 1996
[kinesin]; Veigel et al., 1999
[myosin I]; Sakamoto et al., 2000
[myosin V]; Reck-Peterson et al., 2006
[dynein]). Calculation of the decay coefficient allowed us to estimate a mean PCM run length of 1.9 s (95% confidence interval, 1.6–2.4 s) and mean translocation in a single PCM of 0.49 µm (95% confidence interval, 0.39–0.63 µm), with some motions of >2 µm—traversing almost one fifth of the circumference of a nucleus (~11 µm; ).
To facilitate interpretation of our 3D data recorded at 2-s intervals, we computationally undersampled our higher speed 2D data by considering only every fifth time point. When we overlaid the displacement plot using all frames at 400-ms intervals (, red) on a plot of the steps observed between every fifth frame (2 s; , green), it was clear that PCMs corresponded well with the intermittent large step sizes observed at lower temporal resolution (e.g., ). Indeed, all of the steps of >0.4 µm in 2 s corresponded to a PCM, and thus, the fraction of these large steps in our 3D datasets provides a good measure of the frequency and prevalence of PCMs.
During periods that did not meet the criteria for PCMs, sequential steps exhibited constant changes in direction and limited displacement (e.g., , 24–38 s) and were therefore analyzed in a different way, by considering their cumulative effects over larger time intervals. Plots of the root mean square displacement (MSD; RMSD) provide a direction-independent measure of the distance traveled over all time intervals, minimizing the effects of stochastic changes in direction. The RMSD plot for the PCMs has a steep slope, reflecting the large distances traveled in short periods. Notably, RMSD plots of the non-PCM segments of patch trajectories have a lower slope (). These differences underscore the major contribution made by PCMs to the mobility of patches and to the rate at which they explore the nuclear surface, even though patches are engaged in PCMs only ~15% of the time. Plotting the MSD over time () highlights the mode of movement: objects that move by free diffusion generate linear MSD plots, whereas those undergoing active motion or biased diffusion result in MSD plots with upward curvature (Saxton, 1993
). We detected upward curvature in the MSD plot of PCMs alone, as expected for coordinated motion. However, MSD plots corresponding to all patch motions or non-PCM segments do not show upward curvature, consistent with diffusion being the prevailing mode of motion.
In summary, rapid 2D imaging indicates that the saltatory large steps in our 3D data reflect periods in which chromosome sites associated with the NE translocate processively along linear paths, consistent with motor-driven motion along rigid cytoskeletal elements. Such motions occupy individual patches only a minority of the time but contribute markedly to overall mobility. Analysis of images acquired at high frame rates provides a physical explanation for the variable step sizes seen in our lower resolution data ( and Discussion) and also observed by Baudrimont et al. (2010)
Chromosomes undergo PC-driven motion
To enable analysis of the motion of specific chromosomes, we generated strains expressing HIM-8 fused to GFP (see Materials and methods; hereafter GFP::HIM-8). The fusion protein recapitulated the localization of endogenous HIM-8 (Phillips et al., 2005
), associating with the X chromosome PCs (X PCs) in both premeiotic and meiotic prophase nuclei in the germline (). GFP::HIM-8 was crossed to a strain expressing mCherry::histone H2B (McNally et al., 2006
), which allowed us to assign GFP::HIM-8 foci to specific nuclei, to differentiate premeiotic, TZ, and pachytene nuclei based on chromosome morphology, and to computationally align adjacent time points in time-lapse data (see Materials and methods). In most TZ nuclei, GFP::HIM-8 localized to a single focus, consistent with prior evidence that X chromosome PCs pair rapidly after the onset of meiosis ( and S2
; Phillips et al., 2005
Figure 3. GFP::HIM-8 reveals X PC dynamics. (A) Projection image of 12 optical sections from a single time point showing a field of nuclei expressing GFP::HIM-8 and mCherry::histone. Meiosis progresses from left to right. At the left edge of the field, nuclei have (more ...)
To follow X PCs over time, we collected two-color stacks of optical sections using spinning-disk confocal microscopy. The requirement for multiwavelength 3D imaging reduced our collection rate to 5-s intervals. As elaborated in the next section, these imaging conditions confirmed that X PCs in TZ nuclei undergo PCMs, as expected based on the ZYG-12::GFP imaging (Video 4
). In some experiments, we specifically labeled the X chromosomes in a subset of germline nuclei by injecting fluorescent deoxynucleotides (Jaramillo-Lambert et al., 2007
In TZ nuclei, the PC end of the X chromosome was highly mobile, whereas the distal end of the chromosome remained virtually static throughout data collection ( and Video 5
). Chromosomes exhibited remarkable elasticity: in some nuclei, we observed a transient separation of >2 µm between the bulk of the labeled X chromosome and the focus of GFP::HIM-8 ( and Video 6
). The elasticity of the PC end of the chromosome was also indicated by transient stretching of the GFP::HIM-8 signal to far beyond its normal size. In dramatic examples of this behavior, a bright GFP::HIM-8 focus split into two major foci connected by fainter fluorescence intensity, remaining apart for up to two time points (5 s; and Video 7
). We found that paired GFP::HIM-8 signals, which are normally 0.4–0.8 µm wide, stretched to >1.0 µm in 28% of TZ nuclei within 5 min, indicating that this is a frequent occurrence. We also observed several instances in which paired X PCs fully separated for longer periods of time and then reassociated to form a single focus ( and Video 8
). This suggests that pairing may be stabilized at loci outside of the PC, allowing the PCs to separate without dissociation of the homologues.
These data indicate that chromosomes undergo rapid motions led by their PCs. The viscoelastic properties of meiotic chromosomes apparently allow them to absorb forces along their length such that regions distant from PCs undergo substantially less motion than PCs, even during large PCMs.
PCMs are regulated by meiotic progression but not by pairing
Like endogenous HIM-8 protein, the GFP::HIM-8 reporter localized to X PCs both before and throughout meiotic prophase, enabling us to evaluate PC dynamics as a function of meiotic entry and progression (Video 4 and ). We first compared the dynamics of GFP::HIM-8 foci in TZ nuclei to measurements of ZYG-12::GFP patches at the same stage. Tracking and quantification of X PC motion in TZ nuclei yielded a mean step size of 0.412 µm, corresponding to a mean apparent speed of 0.082 µm/s. Based on the aforementioned analysis for ZYG-12::GFP motion, we defined PCMs as steps of >0.6 µm between 3D images acquired at 5-s intervals. 21.5% of observed steps met this criterion, consistent with the proportion of PCMs observed for ZYG-12::GFP ( and S3
). The duration and velocity of these steps also agrees well with our measurements of ZYG-12 motion, adjusted for the different sampling rate (Fig. S4
). We also acquired single-plane images of GFP::HIM-8 motion at 400-ms intervals to confirm that large step sizes observed at 5-s intervals correspond to PCMs (Fig. S3). Although we note that the mean velocity of X PC PCMs is somewhat higher than the velocity measured for all PCMs using ZYG-12::GFP, the similarity between the data collected for these two reporters indicated that PCMs of X PCs are a good proxy for all PCMs (compare and S3).
Figure 4. PCMs are regulated by meiotic progression but not by pairing. (A) Projections of selected time points showing GFP::HIM-8 and mCherry::histone from single nuclei in the TZ or the premeiotic zone. The left images show complete X PC trajectories overlaid (more ...)
Analysis of GFP::HIM-8 dynamics in premeiotic nuclei, which occupy the region of the gonad distal to the TZ, indicated that PCMs were absent. This was evident from both 3D data acquired at 5-s intervals (; and Video 9
) and 2D images recorded at 400-ms intervals (Figs. S3 and S5
). In premeiotic nuclei, the mean observed step size was 0.195 µm, and only 1.2% of steps traversed >0.6 µm—far below the proportion seen in TZ nuclei ().
Notably, PCs in premeiotic nuclei showed relatively restricted motion within the NE: in TZ nuclei, the mean displacement of PCs over 20 s was 1.2 µm, a magnitude that was never observed over the entire 5-min time course in premeiotic nuclei, which showed a mean displacement of 0.7 µm after 5 min (). This mean displacement of 0.7 µm corresponds to a search area of 1.54 µm2 on the NE, only ~4% of the total area.
The absence of PCMs in the premeiotic zone indicates that PCMs are a regulated aspect of the meiotic program. To further investigate how chromosome dynamics are regulated at meiotic entry, we analyzed X PC motion in the gonads of chk
mutant animals, which fail to initiate many events of the meiotic program: the appearance of patches of SUN-1 and ZYG-12 in the TZ (Penkner et al., 2009
; Sato et al., 2009
), pairing and synapsis, and initiation of recombination (MacQueen and Villeneuve, 2001
). GFP::HIM-8 foci in the TZ region of chk-2
mutants showed similar mobility to those in the premeiotic region of wild-type (WT) animals with respect to step size, displacement, and absence of PCMs (; and S4). PCMs were also absent in gonads of sun-1(jf18)
mutant animals, which have small NE patches and defective pairing and synapsis (; and S4; Penkner et al., 2007
; Sato et al., 2009
). These findings reinforce the idea that aggregation of SUN-1 and ZYG-12 into patches is required for normal early prophase dynamics because HIM-8 is associated with the NE (but not with patches) in WT premeiotic nuclei and during early meiosis in sun-1(jf18)
hermaphrodites (Phillips et al., 2005
; Penkner et al., 2007
; Sato et al., 2009
We compared the dynamics of unpaired HIM-8 foci, which were occasionally observed in early TZ nuclei, to paired foci, and found that the step-size distributions and RMSD plots were indistinguishable (). The fraction of steps of >0.6 µm were 21.5 and 21.6% for unpaired and paired foci, respectively. This result indicates that X chromosome dynamics remain constant throughout the TZ, before and after pairing and synapsis. Moreover, it implies that the forces exerted on PCs are sufficiently strong, or scale proportionately, to be insensitive to the increased drag imposed by association with another chromosome.
Analysis of TZ nuclei with unpaired GFP::HIM-8 signals also allowed us to test whether PCMs are biased in direction such that they directly promote pairing by bringing PCs closer together. Specifically, we tested whether the speed of X PC motion was correlated with the change in distance between homologous foci during the same interval (). This analysis revealed that both smaller fluctuations and PCMs move X PCs away from each other as often as they bring them closer together, consistent with pairing being a consequence of random collisions (see Discussion). This reinforced our conclusion from the ZYG-12::GFP data that individual PCs move independently and nondirectionally during early prophase. It also corroborated our finding that the observed motion is dominated by PC movements along the nuclear surface and is not a consequence of nuclear drift or rotation (Fig. S1).
Upon completion of synapsis, nuclei exit the TZ stage and enter pachytene, which is marked by the dispersal of SUN-1 and ZYG-12 throughout the NE. However, the GFP::HIM-8 reporter remains associated with synapsed X chromosomes and with a single remaining focus of ZYG-12 and SUN-1 (Sato et al., 2009
). Compared with TZ nuclei, X PCs in pachytene nuclei showed markedly reduced motion. This was apparent as a much smaller proportion of steps >0.6 µm and a lower slope of the RMSD plot (; and S4). We also measured the motion of X PCs in syp-1(me17)
mutant hermaphrodites, in which the absence of chromosome synapsis results in an elongated region of leptotene/zygotene stage nuclei, often referred to as an extended TZ (MacQueen et al., 2002
). PC motion in the TZ region of syp-1
mutants was similar to that in WT TZ nuclei (; and S4), consistent with our conclusion that synapsis does not markedly alter chromosome motions. These motions continued with indistinguishable velocities, step sizes, and displacements throughout the extended TZ region (unpublished data). In contrast to a recent study (Baudrimont et al., 2010
), we did not find a significant difference between step sizes in syp-1
mutants compared with WT (P = 0.5, Mann–Whitney U
test). This reinforces the idea that motion is regulated by the meiotic stage of each nucleus and not by extrinsic signals such as its position in the gonad.
PCMs depend on MTs and dynein but not on actin
As described in the aforementioned results and in and , PCMs in TZ nuclei show velocities and a distribution of durations consistent with motor-driven motion along linear cytoskeletal elements. Prior work has shown that disruption of MTs abrogates pairing and synapsis (Sato et al., 2009
). Consistent with these findings, we observed that MT destabilization through injection of 0.1 M colchicine into the gonad eliminated all PCMs, as indicated by a distribution of step sizes similar to premeiotic nuclei (; compare with ) and elimination of most steps >0.6 µm (1.9 vs. 22.1% for buffer-injected control animals; compare with ). In contrast, PCMs were not dependent on actin: animals microinjected with 10 µM latrunculin A exhibited PC motion indistinguishable from control animals, as judged by the step-size distributions, the fraction of steps >0.6 µm (26.2%), and RMSD plots (). Thus, PCMs are completely dependent on MTs but insensitive to destabilization of actin filaments.
Figure 5. PCMs are dependent on MTs but not on actin. (A) Projections of selected time points showing TZ nuclei expressing GFP::HIM-8 and mCherry::histone. X chromosomes are selectively labeled by incorporation of Cy5-dUTP. Animals were injected with colchicine, (more ...)
The activity of cytoplasmic dynein enhances the rate of homologue pairing and is required for synapsis (Sato et al., 2009
). Fluorescently tagged dynein (DHC-1::GFP; Gassmann et al., 2008
) localized to bright, dynamic foci associated with the NE of TZ nuclei (), consistent with immunolocalization of dynein in fixed gonads (Sato et al., 2009
). Importantly, dynein foci were not restricted to patches undergoing PCMs but rather showed similar fluorescence intensity at all patches (Video 10
), indicating that dynein recruitment is not sufficient for PCMs.
Figure 6. Dynein activity is required for PCMs. (A) Maximum intensity projection image from a recording of a hermaphrodite expressing DHC-1::GFP and mCherry::histone. Meiotic progression is from left to right; TZ nuclei show prominent dynein foci at the nuclear (more ...)
We used several approaches to knock down dynein function in the germline. RNAi targeting the dynein light chain gene dlc-1
in animals carrying a temperature-sensitive allele of dynein heavy chain dhc-1(or195)
at a restrictive temperature is an effective way to inhibit dynein activity during meiosis, with only limited mitotic defects (O’Rourke et al., 2007
; Sato et al., 2009
). We found that this double knockdown approach eliminated PCMs (; and Video 10). The step-size distribution in dynein knockdown animals showed a downward shift (), and the fraction of steps >0.6 µm was reduced to 1.2% compared with 10.1% in control knockdowns (); note that PCMs in control animals were also somewhat reduced, likely because of the increased age and/or elevated growth temperature required for this dynein knockdown protocol. High-speed imaging of both GFP::HIM-8 and ZYG-12::GFP confirmed an absence of PCMs (Fig. S5). Similar results were obtained when dhc-1
was knocked down by RNAi in WT animals at 20°C (Fig. S5). Collectively, these data demonstrate that dynein is essential for PCMs, which likely reflect movements along cytoplasmic MTs.
Increased PC mobility in meiotic prophase results from a combination of PCMs and reduced constraints to diffusion
Although PCMs first appear at meiotic entry, concomitant with homology search and the initiation of synapsis, homologue pairing does not require dynein activity (this study; Sato et al., 2009
). We therefore investigated whether other features of early prophase dynamics might contribute to pairing. By comparing plots of RMSD, we asked how extensively PCs explore the nuclear periphery under various conditions. We found that even after dynein knockdown, PCs explore a larger region of the NE in early meiosis than they do in premeiotic nuclei (Fig. S4). This difference can be visualized more directly by overlaying multiple observed trajectories set to initiate at the same point on a sphere (). Trajectories from dynein knockdown recordings display a search radius intermediate between premeiotic and control TZ nuclei. This indicates that the motion of PCs is constrained in premeiotic nuclei and that this constraint is relaxed upon meiotic entry.
Figure 7. Dynein-dependent and -independent chromosome mobility contributes to homologue pairing in early meiotic nuclei. (A) To visualize the search radius of PCs under different conditions, 40 GFP::HIM-8 tracks from WT TZ, WT premeiotic, or TZ in dynein knockdown (more ...)
The increased mobility of PCs upon meiotic entry required both MTs and the activity of the CHK-2 kinase (, , and S4). Prior work has shown that CHK-2 activity is required for SUN-1 phosphorylation and patch formation upon meiotic entry (Penkner et al., 2009
). Interestingly, we found that GFP::HIM-8 foci did show increased mobility upon meiotic entry in animals homozygous for the sun-1(jf18)
allele (Fig. S4), although they did not undergo PCMs. This suggests that the jf18
missense mutation impairs the engagement of PCs with dynein but does not affect the elevated mobility that occurs upon meiotic entry, consistent with the idea that this is promoted by SUN-1 phosphorylation. However, sun-1(jf18)
impairs homologue pairing to a greater degree than dynein knockdown (Penkner et al., 2007
; Sato et al., 2009
), indicating that another activity required for pairing—perhaps the ability of SUN-1 to aggregate—is disrupted in jf18
To understand how these differences in overall mobility might affect PC pairing, we simulated trajectories representing a random walk along the surface of a sphere of 3.5-µm diameter using the step sizes and mean displacements measured for PCs in premeiotic and TZ nuclei (see Materials and methods for details; ). We asked how long it would take for two objects, each 200 nm in diameter, moving along independent trajectories on the same sphere from initial positions one quadrant apart to come into contact (collide; ). Not surprisingly, we found that the time to collision was much lower using the parameters measured for TZ PCs than using premeiotic values: ~6 h was required for 95% of premeiotic simulations to produce a collision versus 18.5 min (0.3 h) for the same fraction of TZ foci to collide (). Elimination of PCMs computationally or experimentally by dynein knockdown resulted in estimates of 43 or 93 min to achieve 95% collision, respectively. Interestingly, the 1.2-h delay in simulated collision time using the dynein knockdown data agrees well with the delay in X chromosome pairing that was estimated based on images of fixed samples (Sato et al., 2009
). Thus, although PCMs facilitate timely pairing and play an essential role in allowing paired chromosomes to synapse (see Discussion), the release of constraints to diffusive chromosome motion strongly contributes to promote homologue pairing during meiotic prophase.