<i>Rabx-5</i> and <i>rabn-5</i> exhibit subtle presynaptic release defects.

<p>Animals placed on 0.5 mM aldicarb, an acetylcholine esterase inhibitor, or levamisole, an acetylcholine receptor antagonist were assayed for movement over time. <i>Rabx-5</i> and <i>rabn-5</i> mutant animals exhibit significant resistance to aldicarb compared to wild type animals<b>.</b> Mean ± SEM, *p<0.05.</p

<i>Rabx-5</i> acts by biasing the cycling state of RAB-5.

<p>A) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(Q78L). The <i>rabx-5</i> mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. B) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(S33N). The <i>rabx-5</i> mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001 C) Fluorescent recovery after photobleaching (FRAP) of YFP::RAB-5 in the synaptic regions of WT, the synaptic and intersynaptic regions of <i>rabx-5,</i> and the synaptic region of <i>unc-16</i> mutant worms in which RAB-5 is biased to the GTP bound state; and FRAP of free GFP. Arrow marks the region of bleaching. Fluorescence recovery after photobleaching of synaptic YFP::RAB-5 in <i>rabx-5</i> mutants is significantly slower than WT but faster than <i>unc-16</i> mutants (p<0.01 at time points from 5–15 sec; mean ± SEM). Intersynaptic YFP::RAB-5 in <i>rabx-5</i> mutant animals exhibits similar dynamics to free GFP.</p

<i>Rabx-5</i> mutant animals age faster and RAB-5 endosomal compartments become disorganized with age.

<p>A) Survival assay indicates that <i>rabx-5</i> mutant animals age faster than wild type animals (log-rank test p<0.0001). B) P_unc-25YFP::RAB-5 expression in the soma and dorsal cord of adult day 1 and day 10 wild-type and <i>rabx-5</i> mutant animals. Synaptic intensity is more variable in adult day 10 animals (F test probability that variances differ p<0.0001). Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively.</p

<i>Rabx-5</i> mutations alter the RAB-5 organization in GABAergic motor neurons.

<p>A) A forward genetic screen was conducted by observing GABAergic motor neurons for changes in localization or intensity of P_unc-25YFP::RAB-5. Initial screening isolated mutants in which YFP::RAB-5 was decreased in the cell soma. Further inspection determined if YFP::RAB-5 was increased at the synapse. B) Gene, transcript, and protein structure of <i>rabx-5</i>. The RABX-5 protein consists of a zinc finger motif (ZF) and a motif interacting with ubiquitin (U) that together regulate association with ubiquitinated endosomal cargo; a membrane binding motif and helical bundle required for association with the endosomal membrane; a Vps9 domain that along with the helical bundle promotes guanine exchange activity; and a coiled-coil region that binds rabaptin-5 and contains a motif for autoinhibition of guanine exchange activity. The <i>rabx-5(qa7800)</i> mutation leads to a truncation at the helical bundle. The <i>rabx-5(tm1512)</i> mutation deletes the start site and the exons encoding the zinc finger motif and the motif interacting with ubiquitn. C) P_unc-25YFP::RAB-5 protein localization in the soma (left column) and dorsal cord (right column) of GABAergic motor neurons in wild type and mutant animals. D) P_unc-25YFP::RAB-5 fluorescence intensity was decreased in the soma of mutant animals and increased in the synaptic and intersynaptic regions. This phenotype is recovered with expression of P_unc-25CFP::RABX-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. **p<0.01, ***p<0.001. E) P_unc-25CFP::RABX-5 expression recovers the <i>rabx-5(qa7800)</i> YFP::RAB-5 phenotype.</p

P_unc-25YFP::RAB-5 localization in mutations of other RAB-5 effectors and VPS9 domain proteins.

<p>A) GABAergic motor neuron cell somas expressing YFP::RAB-5 in animals mutant for the RAB-5 effectors, <i>rabn-5</i> and <i>rabs-5,</i> or mutant for the other VPS9 domain proteins, <i>tag-333</i> and <i>rme-6.</i> B) Quantification of soma YFP::RAB-5 intensity. C) GABAergic motor neuron dorsal cord in these mutant animals. D) Quantification of synaptic intensity. E) Quantification of intersynaptic intensity. <i>Rabx-5</i> works in the same pathway as <i>rabn-5</i> but not <i>rabs-5</i> to regulate RAB-5. <i>Rme-6</i> and <i>tag-333</i> do not exhibit the same phenotype as <i>rabx-5</i>. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region. Bar represents the mean. *p<0.05, ***p<0.001.</p

<i>Rabx-5</i> mutant animals have aberrant synaptic vesicles.

<p>A) P_unc-25Synaptobrevin::GFP intensity is increased in soma and synaptic regions in <i>rabx-5</i> mutant animals. This phenotype is rescued by expression of P_unc-25mCherry::RAB-5(Q78L). B) P_unc-25mCherry::RAB-3 intensity is increased in soma and synaptic regions. C) P_unc-25synaptobrevin::GFP is decreased in soma, synaptic, and intersynaptic regions of larval stage 1 animals. D) YFP::RAB-5 intensity is increased in synaptic regions of the dorsal and ventral cord of larval stage 1 <i>rabx-5</i> mutant animals. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, **p<0.01, ***p<0.001.</p

Operation of ALCATRAS.

<p>A, B) Schematics showing the removal of a daughter cell by the media flow when the mother buds at the top of the trap (A) or at the bottom (B). In both cases the flow is from top to bottom (red arrow). The newly formed daughter cells follow the streamlines shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100042#pone.0100042.s001" target="_blank">Fig. S1A</a>. C) Microscopy images of removal of daughter cells in the device. The cells are expressing Doa1p-GFP, and the fluorescence image has been overlaid with the DIC image for clarity. Scale bar indicates 5 µm. D) Success rates for four ALCATRAS experiments. The number of cells retained in their original traps over the time course is plotted for two independent experiments using each of ALCATRAS 1 (red – more dense spacing of traps) or ALCATRAS 2 (blue – less dense spacing of traps). Only cells that were present in the traps at the first time point are included. Results were scored manually from a random subset of the fields imaged. Numbers include cells that have visibly died during the experiment. For a more detailed breakdown of cell loss and replacement, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100042#pone.0100042.s003" target="_blank">Fig. S3</a>. Flow rates were 2 µl/min from each input syringe pump (4 µl/min total). E) Cell viability plotted as a function of the number of replications (n = 422). Cells were observed for 62 hours, and replicative lifespans were scored manually. The mean lifespan is 22.4. Flow rates were 2 µl/min from each input syringe pump (4 µl/min total). F) Kymograph showing Hsp104-GFP expression over time with imaging every 10 mins. The median fluorescence intensity within the area of each cell (n = 1003) at each time point is shown by the colour map. Only cells that are present during the first hour of the experiment and that remain in their original traps for at least 10 hours are shown.</p

Overview of ALCATRAS.

<p>A) A DIC image of the cell traps. The cells are expressing Doa1p-GFP, and the fluorescence image has been overlaid with the DIC image for clarity. Traps consist of two vertical pillars that trap mother cells and allow daughters to flow away. The small size of the traps permits a high density of cells in the field of view (more than 40 mother cells can be imaged at 60x magnification in ALCATRAS 1). B) A schematic depicting fluid flow in the flow cell during media switching. Switching occurs within six seconds by changing the flow rate of syringe pumps. C) Overview of the microfluidic device. Each device contains more than 1500 individual traps. D) The switching rate and reliability of media switching in traps. Media switching was assayed using 0.1% fluorescein in one of the two media. E) Schematic showing trap dimensions in microns.</p

Cell cycle dynamics in ALCATRAS.

<p>A) Frames from a time-lapse movie showing a cell expressing Whi5p-GFP across one cell cycle. Fluorescence is localized in the nucleus during late M and early G1 phases. B) Plot of the nuclear localization of Whi5p over time for the cell shown in A. Frames in A are from the shaded period. Over many hours the cell undergoes a large number of cell cycles, resulting in a strongly periodic signal. The inset shows the power spectrum derived from applying Welch’s windowing algorithm and the Fourier transform to this data with a single peak at the frequency of the cell cycle. C) Histogram showing the distribution of cell cycle times of mother cells undergoing their first three divisions in ALCATRAS. D) Kymograph illustrating the change in replicative age of individual cells during the experiment. To aid visualization of the cell cycle, each alternate division is marked by a different colour. The total number of divisions undergone in the device when odd is shown by the colour map; even numbers of division are depicted in dark blue. Cells have been ordered by their number of divisions, and only cells that remain alive and in their original traps throughout are illustrated (299 cells). Flow rates were 2 µl/min from each input syringe pump (4 µl/min total).</p

Device comparison.

<p>A comparison of selected commercial and academic devices that have demonstrated single-cell time-lapse cultures for budding yeast. ¹ Numbers reported refer to the original, cited papers and represent reported data, not theoretical maximum numbers. Some devices, such as the Cellasic, have many citations in which larger numbers of cells may have been recorded and for longer periods. The number of cells refers to those cells originally loaded and excludes daughter cells born in the device except for the Microfluidic imaging matrix and Microchemostat arrays², for which numbers of loaded cells were not reported. Note the trade off between the intervals between imaging frames and the number of cells recorded, and that the density of cells in view has not been reported for most devices. Note also that larger cell numbers can be imaged for shorter times in devices that suffer loss of mother cells over time, such as the ALCATRAS devices. We have assumed that all reported datasets come from a single experiment with a single device.</p