Traffic 2004; 5: 544–559 Blackwell Munksgaard
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Blackwell Munksgaard 2004
doi: 10.1111/j.1600-0854.2004.00195.x
Dense Core Vesicle Dynamics in Caenorhabditis elegans Neurons and the Role of Kinesin UNC-104 Tobias R. Zahn1,2,§, Joseph K. Angleson3, Margaret A. MacMorris4, Erin Domke1, John F. Hutton1,*, Cindi Schwartz5 and John C. Hutton1
Received 27 October 2003, revised and accepted for publication 21 April 2004
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The regulated exocytosis of polypeptide hormones and neuropeptides stored within dense core vesicles (DCVs) forms the cell biological mechanism of peptidergic neurotransmission and much of the endocrine function in metazoans. While the exocytosis of DCVs and synaptic vesicles (SVs) share common molecular machinery, there are fundamental differences in the biogenesis of these organelles and their trafficking within the cell. SVs undergo multiple rounds of exocytosis and endocytosis within the synaptic terminal and are equipped with enzymes and transporters necessary to generate and replenish their cargo of classical neurotransmitters such as acetylcholine (1). By contrast, DCVs bud from the trans-Golgi network within the perinuclear region of the cell body and are dependent upon de novo biosynthesis of their cargo proteins and accessory enzymes (2). Following neuropeptide secretion, DCV membrane constituents are thought to be reutilized (3,4) and, accordingly, DCV function is dependent on efficient long-range transport in both anterograde and retrograde directions. DCVs are subject to microtubule (MT)-based fast neuronal transport (5,6), as is the case for synaptic vesicle precursors and other small vesiculotubular membranous organelles in neurons (6,7). Long-range DCV transport therefore is thought to be mediated by motor proteins of the large kinesin and dynein superfamilies (8).
Barbara Davis Center for Childhood Diabetes and Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA, 2 Institut fu¨r Zellbiologie, Universita¨t Witten/Herdecke, 58448 Witten, Germany, 3 Department of Biological Sciences, University of Denver, Denver, CO 80208, USA, 5 Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA §Present address: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. *Corresponding author: John C. Hutton, PhD,
[email protected] 4
We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)-tagged IDA-1. IDA-1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA-1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron-dense objects in the neuronal processes. The post-translational processing of IDA-1::GFP in transgenic worms was dependent on the neuropeptide proprotein convertase EGL-3, indicating that the protein was efficiently targeted to the peptidergic secretory pathway. Time-lapse epifluorescence microscopy of IDA1::GFP revealed that DCVs moved in a saltatory and bidirectional manner. DCV velocity profiles exhibited multiple distinct peaks, suggesting the participation of multiple molecular motors with distinct properties. Differences between velocity profiles for axonal and dendritic processes furthermore suggested a polarized distribution of the molecular transport machinery. Study of a number of candidate mutants identified the kinesin UNC-104 (KIF1A) as the microtubule motor that is specifically responsible for anterograde axonal transport of DCVs at velocities of 1.6 mm/s2.7 mm/s. Key words: axonal transport, cell polarity, IA-2, immunoelectron microscopy, secretory granules, UNC104, video microscopy
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A large number of genes encoding MT-based molecular motors have been identified, with at least 50 kinesins and three dyneins in mammals and 22 kinesins and two dyneins in Caenorhabditis elegans (9,10). Kinesins form a heterogeneous family characterized by a conserved motor domain. Force generation from MT-stimulated adenosine 5’-triphosphate (ATP) hydrolysis is turned into processive unidirectional movement along the MT (plus-end directed for most kinesins) aided by dimerization of neck and stalk domains. A divergent tail domain is responsible for cargo binding (11). In neurons, the conserved ‘monomeric’ kinesin UNC-104/KIF1A (12,13) is an essential motor for axonal transport of SV precursors to the synapse (14,15). Mechanisms of peptidergic neurosecretion show considerable conservation between C. elegans and mammals, as indicated for instance by the use of post-translational endoproteolysis as a mechanism to excise peptidergic
IDA-1 :: GFP Transport in C. elegans Neurons
neurotransmitters from larger proprotein precursors. This is reflected at the molecular level in the conservation of genes such as the proprotein convertase (PC) EGL-3 (16), its cofactor 7B2 (T03D8.3) (17), carboxypeptidase EGL-21 (18), and neuropeptides of the insulin family (19). C. elegans IDA-1 (related to islet cell diabetes autoantigen) represents the single ortholog (20,21) of the two closely related type 1 transmembrane proteins ICA512 (22) (also known as IA-2) and phogrin (23) (IA-2b) which form a catalytically inactive subfamily of receptor-type protein tyrosine phosphatases localized to DCVs of mammalian neuronal and neuroendocrine cells (23–25). Phogrin–GFP (green fluorescent protein) fusion proteins have been used successfully to visualize DCVs in mammalian cell lines (3,26,27), and recent studies with GFP-tagged molecular motors and intraflagellar transport proteins have demonstrated the feasibility of visualizing neuronal transport within C. elegans neuronal processes in vivo (28–31). We previously reported that IDA-1 is expressed in a subset of about 10% of the C. elegans hermaphrodite 302 neurons (20). Here we show that IDA-1 and an IDA-1::GFP fusion protein are localized to DCVs like their mammalian counterparts and that the latter provide a useful marker tool to study the real time movements of DCVs in C. elegans neuronal processes. Velocities of IDA-1::GFP neuronal transport reached 4 mm/s and clustered at specific rates. They differed for retrograde and anterograde transport within the same neuronal process and furthermore in opposing processes of the same neuron, demonstrating a polarized distribution of the transport machinery. Study of IDA-1::GFP transport in unc-104 mutants revealed that kinesin UNC-104 is specifically responsible for its anterograde transport in axonal processes at velocities of 1.6–2.7 mm/s. Concomitantly unc-104 mutant animals showed an increase in IDA-1::GFP fluorescence in cell bodies and reduction in axonal termini. Similar macroscopic changes in DCV distribution could provide the basis for future genetic screenings for molecules involved in DCV transport and its regulation.
that are predicted to be subject to post-translational proteolytic processing within the DCVs by members of the Kex2-like proprotein convertase (PC) family. Western blotting of IDA-1::GFP transgenic nematode homogenates with an anti-GFP antibody showed the presence of 140 kDa, 95 kDa and 85 kDa forms of the fusion protein (Figure 1), sizes consistent with a precursor (predicted Mr 115 kDa) and a mature protein (72 kDa) generated by post-translational endoproteolytic cleavage at two of several potential dibasic consensus sequences such as KRRK367 in the lumenal domain of the protein (20), similar to the processing of ICA512 (25). Western blot analysis of transgenic animals carrying a mutation in the egl-3 gene showed the 140 kDa band of the precursor form, but not the 85 kDa product, and reduced presence of the 95 kDa form. EGL-3 (KPC-2) represents the single C. elegans ortholog of the mammalian neuroendocrine PC2 and PC1 (16,32) and is present in all ida-1 expressing cells (20). It has been shown to be required for the processing of FMRFamide-related neuropeptides (18) and thus appears to be the functional homolog of those mammalian PCs. Western blotting with an anti-IDA-1 cytosolic domain antibody showed the presence of the 140 kDa precursor in IDA-1::GFP transgenic worms as expected and the processed 85 kDa product in wild-type but not egl-3 mutants (Figure 1). These analyses also showed the presence of
Results IDA-1::GFP transgene expression and proteolytic processing by EGL-3 Transgenic nematodes expressing a full-length IDA-1::GFP fusion construct under the control of the ida-1 gene promoter were generated for in vivo microscopy studies. The predicted membrane topology of IDA-1::GFP is equivalent to that of phogrin–GFP used in previous studies of vesicle transport in mammalian neuroendocrine cells (26) and locates the C-terminal GFP-tag in the cytoplasm. The ‘lumenal’ domain of IDA-1, like phogrin (23) and ICA512 (25), has two sites marked by pairs of basic amino acids Traffic 2004; 5: 544–559
Figure 1: Western blot of C. elegans extracts from wild-type (N2) worms and egl-3 mutant worms expressing IDA-1::GFP as indicated and developed with antibodies raised to GFP or the IDA-1 cytosolic domain. The mobilities of the IDA-1::GFP precursor (140 kDa) and processed forms (85 kDa, 95 kDa) are marked with arrows, the endogenous IDA-1 precursor (95 kDa) and processed forms are marked by dots.
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a 95 kDa protein that is consistent with the endogenous IDA-1 (predicted Mr 87 kDa) and a diffuse band running close to the gel front that could represent processed products (predicted 48 kDa for KRRK367). As in the case of IDA-1::GFP transgenic nematodes, the formation of the processed products was not evident in egl-3 mutants. These analyses indicated that introduction of the IDA1::GFP transgene under the ida-1 gene promoter resulted in net increase in the overall expression of the IDA-1 molecule of approximately 2- to 3-fold. Processing of the transgene product under steady state conditions was incomplete at 37 7% of the total (n ¼ 4) as determined by densitometric analysis of blots developed with either anti-GFP or anti-IDA-1 antibodies but was similar to that of the endogenous IDA-1 (32–46%, n ¼ 2). The subcellular distribution of the pro- and mature forms was not investigated but conceivably reflects the relative distribution between the cell body and neuronal processes. Whatever the case, the data are consistent with the observation that IDA-1 and EGL-3 share a common subcellular localization in the regulated secretory pathway of the nematode during the biogenesis of the organelle and that neither the level of expression nor the presence of the GFP fusion marker have affected the targeting of the molecule to the secretory pathway.
IDA-1 immunolocalization Immunofluorescence microscopy of wild-type hermaphrodites using anti-IDA-1 antibodies (Figure 2) revealed strong immunoreactivity within the pharyngeal nerve ring (Figure 2A,D, and Supplement 1 available on-line at http:// www.traffic.dk/suppmat/5_6.asp), around the vulva (Figure 2A,B) and the tail (Figure 2A,G,H), and variable staining at the nose tip (Figure 2A,D). Males showed weaker staining of the nerve ring and intense staining of male-specific tail neurons (Figure 2C and cover illustration). Immunoreactivity was detectable from the L1 larval stage onwards. The observed cellular expression pattern matches that of a transcriptional pida-1::GFP reporter construct (20). At the subcellular level, the antigen was localized to cell bodies exterior to the nucleus and to neuronal processes (Figure 2D–H). These included amphid sensory dendrites (Figure 2D), ALA lateral processes (Figure 2E), processes of the ventral and dorsal nerve cords (Figure 2F) and PHC neurons in the tail spike (Figures 2G,H). Staining along the processes was punctate in appearance and distributed among a large number of small individual elements and a few brighter, apparently larger objects. The punctate distribution is consistent with a vesicular expression pattern as observed in the mammalian homologs of this protein. The subcellular localization of IDA-1 differed from that of SV proteins reported in C. elegans. The latter are found in all neurons, where they are localized to a limited number of large clusters corresponding to presynaptic sites. Unlike IDA-1 immunoreactivity, they are notably absent in cell bodies and amphid dendrites (33). 546
IDA-1::GFP transgene localization Epifluorescence microscopy revealed IDA-1::GFP expression in a subset of the cells previously shown to transcribe the ida-1 gene (20) (Figure 3; see Supplement 2 available on-line at http://www.traffic.dk/suppmat/5_6.asp for broad perspective). Expression was prominent in the ALA neuron in the dorsal ganglion of the pharyngeal nerve ring (Figure 3B), the hermaphrodite-specific vulval motoneurons HSN, VC4 and VC5, the neuroendocrine-like uv1 cells, the tail neuron pair PHC (Figure 3C), was also present in the ventral cord neurons VC1, VC2, VC3, VC6, and was somewhat variable in a few amphid neurons. Cytoplasmic GFP fluorescence was high in cell bodies and extended into the nerve processes in a distinct punctate pattern. The subcellular distribution of IDA-1::GFP was similar to that of IDA-1 as observed by immunofluorescence microscopy of wild-type animals (Figure 2), as best observed in the ALA lateral process and PHC (Figures 2–4). Ultrastructural studies of IDA-1::GFP transgenic animals were performed on uv1 neuroendocrine cells in the vulval region (data not shown) and the processes emanating from the ALA cell body in the pharyngeal nerve ring (Figure 4). The latter analysis required locating and sectioning single cells whose cell bodies are less than 10 mm in diameter and whose processes, although long, are less than 1 mm in diameter. Two such processes were found in the expected position adjacent to the dorsal surface of the isthmus of the pharynx (Figure 4). The processes contained aggregates of electron-dense material that were immunoreactive to GFP antibodies as revealed by immunogold staining. The labeling index of a 50 nm diameter region surrounding each density (156 14 particles/mm2, n ¼ 200 observations) was significantly greater (p < 0.00001) than that of the surrounding cytoplasm (8.9 3.0, n ¼ 110) and adjacent mitochondria (6.9 5.0, n ¼ 72) in the same cells and the labeling index of adjacent neuronal processes (11.0 5.0, n ¼ 108). A similar concentration of GFP immunoreactivity was associated with spherical electron-dense objects of 50 nm diameter in uv1 cells (data not shown). Fast time-lapse microscopy of live IDA-1::GFP transgenic animals revealed that the small IDA-1::GFP punctae were mobile, further supporting a vesicular localization (Figure 3D, Supplements 3, 4 available on-line at http://www.traffic.dk/suppmat/5_6.asp). The apparent size of the GFP punctae was below the spatial resolution limit of light microscopy ( 250 nm). This is consistent with their identity with the immunoreactive electron-dense structures observed above and the reported existence of DCVs in C. elegans neurons of 37–53 nm diameter in electron micrographs of osmium-fixed nematodes (34). Brighter and larger punctae appeared to consist of local accumulations of individual smaller punctae, as in many cases they were observed to disperse within minutes. Composite images of entire ALA neurites were obtained at 1-h intervals for 3 h (Figure 3B) to assess the stability of the preparation. These showed around 1000 discrete punctae, distributed uniformly along Traffic 2004; 5: 544–559
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Figure 2: IDA-1 immunostaining in wild-type C. elegans showing punctate staining in a subset of neurons. Anterior to the left. A) Adult hermaphrodite. B) Hermaphrodite vulva. The morphology appears collapsed due to the fixation procedure. C) Male tail showing strongly immunoreactive neurons of the copulatory apparatus. D) Head from dorsal perspective showing pharyngeal neuronal cell bodies (arrows), their anterior processes (small arrowheads) and the amphid ciliated endings (large arrowheads). E) ALA lateral neuronal process that has numerous in-focus punctae varying in brightness (arrowheads). F) VC2 neuron (arrow) in the right ventral nerve cord. Arrowhead indicates processes in the left ventral nerve cord. G) PHC cell body with an in-focus region of the posterior process to the right showing punctate staining of varying intensity (arrowheads). H) PHC posterior process in the tail spike showing punctate staining (arrowheads) approximately 100 mm posterior to the cell body.
the lateral process at each time point. Around 20 of the larger fluorescent objects were at invariant positions (Figure 3B) and in some cases corresponded to the approximate locations of synapses (34), notably in the pharyngeal Traffic 2004; 5: 544–559
nerve ring (Figures 3A,B1 and 4B) and the extreme tip of the neuronal process adjacent to the anus (Figure 3B6). A second class of apparently static objects appeared to change in fluorescence intensity over this time period (Figure 3B). 547
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Figure 3: IDA-1::GFP localization by fluorescence microscopy in living nematodes. A) Cartoon depicting the expression pattern in an adult hermaphrodite. Anterior to the left, lateral view. IDA-1::GFP expressing neurons and their processes are indicated and the cell bodies of ALA and PHC are marked with arrows (see Supplement 2 available on-line at http://www.traffic.dk/suppmat/5_6.asp for complete worm image). The lower case letters indicate regions used for time-lapse microscopy and correspond to the panels of Figure 7. B) The fluorescence pattern in a left ALA lateral process is traced from its point of exit from the cell body in the head to a terminal synapse near the anus in the tail. Numerous punctate bodies are visible along the neurite; larger brighter fluorescent regions that were stable and immobile over the course of 2 h are marked by solid arrowheads; open arrowheads indicate areas of bright fluorescence that changed in intensity with time. C) Punctate IDA-1::GFP pattern in the left PHC cell body and proximal part of the posteriorly directed process. D) Path of an IDA-1::GFP-tagged vesicle moving from a perinuclear region into the posterior process (see Supplement 3 for video, available on-line at http://www.traffic.dk/ suppmat/5_6.asp).
Genetic analysis of intracellular IDA-1::GFP distribution To further define similarities and differences between SVs and IDA-1::GFP-labeled vesicles, IDA-1::GFP transgene expression was analyzed in various mutant backgrounds 548
known to disrupt SV localization. Mutations in sad-1 (35), syd-2 (36), rpm-1 (37,38), and unc-51 (35,39) result in aberrant synaptic morphology. In unc-11 mutants the SV membrane marker synaptobrevin/VAMP SNB-1 was reported to be mislocalized, diffusely staining the plasma Traffic 2004; 5: 544–559
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Figure 4: Immuno-electron microscopy of IDA-1::GFP transgenic C. elegans. A) Wide-field epifluorescence micrograph superimposed on brightfield image (anterior to left, ventrolateral perspective). Two axonal processes emerge from the single ALA cell body (arrow) located in the dorsal ganglion above the isthmus of the pharynx. The processes curve anteriorally and ventrally over the musculature of the pharynx and then posteriorly in an equatorial position beneath the hypodermal ridge. B) Laser scanning confocal micrograph of a nematode optically rotated to the equivalent position as the animal in panel A showing cell body (arrow) and multiple fluorescent punctae, a number of which occupy the same location as in animal A. Some detail in the right (lower) axon is lost because of the thickness of the specimen. The line indicates the approximate plane of section of the electron micrographs shown in panels C–E. C) Low-power electron micrograph montage of an immunostained transverse section through the isthmus of the pharynx. The central dark structure is the pharyngeal lumen containing E. coli (which also adhere to the cuticle of the nematode). The pharyngeal nerve ring envelops the musculature surrounding the lumen and comprises isolated neuronal cell bodies (arrows) and many neuronal processes of up to 1 mm diameter which are irregular in section. D, E) Serial sections of the nerve ring 0.1 mm and 0.2 mm anterior to the boxed region in panel C showing two neuronal processes containing circular regions of electron density that are immunoreactive with antibodies directed to GFP (10 nm gold particles). Immunoreactivity appears confined to these two processes and not to other processes in the nerve ring or other organelles or tissue.
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membrane of axons as well as cell bodies and dendrites, which was attributed to a defect in endocytosis (28,40). No effect on subcellular IDA-1::GFP distribution was apparent in rpm-1, sad-1, and unc-11 mutants. In unc-51 and syd-2 mutants, which have enlarged synapses (35,36), some neuronal processes displayed a few large clusters of fluorescence corresponding to reported locations of synapses (e.g. ALA lateral processes near the cell body, PHC anterior processes in the ventral nerve cord) (34); however, the overall appearance of neuronal processes with punctate IDA-1::GFP staining was not affected. Neuronal kinesin UNC-104/KIF1A plays a role in axonal anterograde transport of SV precursors from the cell body to the synapse in both mice (15) and worms (14,33) and more recently was also shown to be involved in the transport of C. elegans neuropeptides and their processing enzymes (18). In unc-104(rh43) and unc-104(e1265) mutant animals, IDA-1::GFP fluorescence intensity was significantly increased in cell bodies relative to the ALA lateral processes and ALA neurite tip (Figure 5). The PHC neurons were affected to a lesser extent.
No effect on the intracellular distribution of IDA-1::GFP was detected in a series of candidate mutants affecting molecular components of the exocytosis/endocytosis machinery. These included aex-3 (41), cab-1 (42), rab-3 (42,43), synaptobrevin snb-1 (44), syntaxin unc-64 (45,46), synaptotagmin snt-1 (47), unc-13 (48), CAPS unc31 (49), and rme-1 (50). Also, no effect was seen in the unc-51-interacting unc-14 (51) and PC egl-3 (16).
Trafficking of IDA-1::GFP Using time-lapse microscopy, IDA-1::GFP tagging allowed us to observe in real time transport events of vesicle carriers with a presumed function in neuropeptide secretion. The restricted cellular expression of IDA-1::GFP facilitated observation of vesicle behavior due to the lack of fluorescence contribution from neighboring cells. The thin ( 250 nm diameter) and rarely branching neuronal processes of C. elegans permitted the tracing of vesicle movements over distances in the order of 50 mm, and time-lapse image acquisition with a sensitive CCD-camera could be performed at rates of 5 Hz for several minutes before photobleaching became limiting.
Figure 5: IDA-1::GFP expression in adult hermaphrodite C. elegans wild-type (A–E) and unc-104(rh43) (F–I) animals. Unc-104 mutants have a dumpy phenotype. IDA-1::GFP is accumulated in ALA cell bodies (arrows) relative to lateral process termini (arrowheads), whereas the relative distribution in the PHC cell body and posterior processes are not significantly affected.
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The linear nature of the neuronal processes permitted the convenient transformation of time-lapse recordings into kymographs (52) for quantitative analysis (Figure 6 and Table 1). In such representations, stationary vesicles appear as horizontal traces and moving vesicles as diagonals whose slope is proportionate to object velocity (Figure 6B). As the fluorescence of an individual puncta appears brightest at rest and diminishes with increasing velocity because individual pixel elements of the CCD camera chip will be exposed to the fluorescence signal from a moving object for a proportionally shorter period of time, it is important to note that IDA-1::GFP fluorescence provided a robust signal of sufficient strength to reliably detect even fast velocities in our assay conditions (Figure 6). The small IDA-1::GFP punctae moved in both anterograde and retrograde directions relative to the cell body. Fast long-range transport and short-range oscillatory motions were observed as well as bidirectional and saltatory movements (Figure 6; Supplements 3, 4, available on-line at http://www.traffic.dk/suppmat/5_6.asp) similar to those reported with phogrin–GFP-tagged DCVs in mammalian cells (26,27) and characteristic of MT-based fast axonal transport (53,54). IDA-1::GFP moved at velocities of up to 4 mm/s (Figure 6), which was similar in magnitude to MTbased fast neuronal transport in a range of organisms (8) including C. elegans (28,30,31). All movement could be blocked by exposure of animals to sodium azide or nocodazole, suggesting that transport was ATP- and MT-dependent, respectively. Table 1 summarizes data from 18 representative recordings from ALA lateral processes and 20 recordings from PHC posterior processes. The results for run lengths and frequencies of specific behaviors such as reversals can only provide lower estimates. The calculated values depend to a large degree on the duration of video observations and the length of the neuronal process captured in the focal plane of the objective. The simultaneous transport of several vesicles in close proximity was frequently observed, which appeared as a single fluorescent object due to their small size (Figure 6, Table 1).
Velocities of IDA-1::GFP-tagged vesicle transport Velocity analyses of the movements of IDA-1::GFP-tagged vesicles displaying fast (> 0.5 mm/s) directed transport were performed on the processes of the PHC, ALA, VC6, and amphid neurons (Figure 7). In the posteriorly directed processes of the PHC neurons which extend into the tail tip, anterograde velocities clustered at distinct values and reached as high as 3.0 mm/s (Figure 7A). A KolmogorovSmirnov statistical test suggested that this velocity distribution was non-normal (p ¼ 0.012). Least-square curve fitting analysis with three Gaussian peaks calculated the velocity peak positions as 0.97 0.07 mm/s, 1.53 0.02 mm/s, and 2.56 0.04 mm/s. By contrast, retrograde movements in the same neuronal process did not exceed 1.5 mm/s and were well fitted by two peaks at 0.66 0.01 mm/s and 0.95 0.01 mm/s (Figure 7A). In the Traffic 2004; 5: 544–559
Figure 6: Movement of IDA-1::GFP-tagged vesicles. A) A timelapse series of 160 frames at 201 ms intervals was acquired from a PHC posterior process (see Supplement 4 for movie, available on-line at http://www.traffic.dk/suppmat/5_6.asp). Five representative frames separated by 8 s are shown. Anterograde movement is upwards. B) Kymograph of the time-lapse series represented in A. Fluorescence intensity along the vertical axis is plotted vs. time. Arrows indicate examples of fast, long-range transport, large arrowheads random, jittering movement, and small arrowheads immobile vesicles. Other characteristic densecore vesicle movements include: C) Fast, directed movement interrupted by brief pauses (arrows); D) Apparent displacement of resting vesicles by a fast moving passing vesicle (arrows); E) Synchronous movement of vesicles in close proximity as evidenced by bifurcating tracks (horizontal arrow); F) Synchronous movements of distinct vesicles spaced 500 nm apart. Some movement of the worm is also evident (faded area); G) Abrupt reversal of direction; H) Movement of several vesicles disrupted in the same location (arrows).
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Zahn et al. Table 1: Quantitation of DCV movements in ALA lateral processes (n ¼ 18 recordings) and PHC posterior processes (n ¼ 20). ‘Objects’ are here defined as mobile discrete fluorescent punctae, and do not necessarily represents single vesicles as evidenced by ‘fusion’ or ‘fission’ events that appear to involve collections of vesicles. Movements are frequently interrupted by pauses, and runs are defined as object movements of at least 0.5 mm uninterrupted by cessation of movement for > 500 ms. The calculated run lengths and frequencies of events (e.g. reversals) should be considered a lower estimate since the beginning or end of longer runs was not always captured within the time period of observation or because an object moved out of the field of view Neuronal process Average video duration [s] Average video length [mm] Number of moving objects Objects with reversals Objects with ‘fusion’ or ‘fission’ event Objects with a separate object in close proximity moving at the same velocity Direction of movement Number of runs Average run length [mm] Runs longer than 2 mm Runs > 2 mm with velocity changes Runs > 2 mm not captured in their entirety
long posteriorly directed processes of the ALA neuron, which run laterally from the pharyngeal nerve ring to the preanal ganglion, anterograde movement displayed a similar velocity range to that in PHC posterior processes (Figure 7F). The retrograde transport rates in this case attained speeds of 4 mm/s; 84% (122/145) of the recorded velocities exceeded 1.5 mm/s (Figure 7F). Neuronal polarity of IDA-1::GFP transport velocities The observation of differences in the profiles of retrograde transport velocities between the PHC posterior and ALA processes (Figure 7A,F) raised the question of whether each neuron or neuronal process had a unique velocity profile. Two other velocity histograms recorded from VC6 anterior processes (Figure 7C) and amphid sensory dendrites (Figure 7E) were shaped similarly to that of the PHC posterior processes (Figure 7A). They shared the major velocity clusters near 0.7 mm/s and 1.0 mm/s in retrograde direction and near 1.0 mm/s, 1.5 mm/s and 2.6 mm/s for anterograde movements. The velocity profiles for PHC anterior processes (Figure 7B) and VC6 posterior processes (Figure 7D) more closely resembled that of the ALA lateral processes (Figure 7F) by virtue of the predominance of retrograde transport at rates faster than 1.5 mm/s. This apparent dichotomy may reflect the axonal or dendritic nature of the nerve processes: The two long bilaterally symmetrical posteriorly directed sublateral processes of the ALA neuron terminate with synapses onto PVC (34) and thus have axonal characteristics. PHC is a morphologically polarized neuron with an anterior process with axonal characteristics and a posteriorly directed dendritic process (34). The PHC axonal process was difficult to image as it curves out of the focal plane, nevertheless a sufficient number of observations was made to conclude that retrograde movements > 1.5 mm/s were prominent (Figure 7B). Amphid neurons are also clearly polarized with axonal projections in the nerve ring and dendrites 552
ALA lateral process
PHC posterior process
44.5 14.2 36.3 8.1 104 9 23 6 anterograde retrograde 88 68 3.1 2.5 6.3 7.0 48 47 12 18 22 31
18.8 6.0 36.9 16.8 106 28 37 11 anterograde retrograde 78 79 6.6 7.1 2.8 2.7 55 36 30 16 37 20
extending to the tip of the nose where they terminate in ciliated sensory endings (28,34,55). The velocity profile here showed slower retrograde transport (Figure 7E) similar to the PHC posterior process (Figure 7A), suggesting that in polarized C. elegans neurons dendrites generally use slower retrograde transport and axons use faster. In spite of the lack of morphologic polarization in the VC6 neuron (34), the observed vesicle velocities in each of its arms were different (Figures 7C,D), suggesting that neuronal polarity of the transport machinery existed at the molecular level. Anterograde transport is impaired in unc-104 kinesin mutant animals The observed accumulation of IDA-1::GFP in cell bodies of unc-104 mutants (Figure 5) suggested that UNC-104 (KIF1A) represents an anterograde motor of IDA-1::GFP transport. Velocity analysis was performed in the ALA lateral process and PHC posterior process representing the two different classes of velocity profiles described above (Figure 7). IDA-1::GFP transport velocities in unc104(e1265) mutants revealed that velocities in PHC posterior processes (Figure 8A) and retrograde transport velocities in ALA lateral processes (Figure 8B) appeared very similar to those of wild-type animals. Anterograde transport in ALA lateral processes (Figure 8B), however, showed only two out of 55 objects (4%) moving faster than 1.6 mm/s. Most of anterograde transport in ALA lateral processes of wild-type animals (Figure 7F) occurred at velocities >1.6 mm/s (101/176 or 57% of observations), implying that those represent the inherent in vivo velocities of UNC-104 kinesin when transporting IDA-1::GFPtagged vesicles and that UNC-104 makes at best minor contributions to IDA-1::GFP transport at velocities slower than 1.6 mm/s. It also follows that at least one other motor contributes to anterograde IDA-1::GFP transport in ALA lateral processes at velocities up to 1.9 mm/s. The loss of Traffic 2004; 5: 544–559
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Figure 7: Vesicle transport velocity profiles in various neuronal processes. Velocities were plotted with a bin width of 0.1 mm/s, with anterograde movements positive and retrograde negative. Each observation represents the sustained movement of a fluorescent object at constant velocity (see Materials and Methods for detail). The locations of the neuronal processes analyzed are indicated in Figure 3A. Two distinct types of velocity profiles are evident, indicating neuronal polarity. Axonal profiles are grouped in the right column, dendritic in the left. A) Gaussian curves from a best fit are shown in red.
Figure 8: Vesicle transport velocities in PHC posterior processes (A) and ALA lateral processes (B) in unc-104(e1265) kinesin mutants. Anterograde velocities in ALA lateral processes faster than 1.9 mm/s are absent.
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the high velocity anterograde component in mutant animals is reflected in a highly significant decrease in the mean anterograde velocity and there is a corresponding, but not significant, decrease in the number of punctae moving in an anterograde direction in ALA processes that is consistent with a reduction in fluorescence at the tip of this process (Figure 5).
Discussion Subcellular localization of IDA-1::GFP IDA-1 represents the single C. elegans ortholog of the mammalian ICA512/phogrin gene family (20,21) and, like its mammalian orthologs, IDA-1 appears to be localized to DCVs. Unlike SVs, IDA-1 and IDA-1::GFP were not restricted to presynaptic sites but were abundantly localized throughout cell bodies, axons and dendrites of expressing neurons. IDA-1::GFP post-translational processing was dependent on EGL-3, the C. elegans ortholog of the mammalian DCV lumenal enzymes PC2 and PC1, which is required for the generation of FMRFamide-related neuropeptides in the nematode (18). IDA-1 is apparently not an obligatory component of all DCVs since it is not expressed in all neurons with ultrastructurally identifiable DCVs (34) nor did it occur in all neurons expressing neuropeptides (56,57). A similar situation prevails with respect to the cellular and subcellular localization of the mammalian counterparts ICA512 and phogrin in mammalian brain (20,25). The current data on IDA-1 immunolocalization, the subcellular localization of IDA-1::GFP and locomotory behavior of IDA-1::GFP vesicles show strong parallels to observations with phogrin and ICA512 in mammalian tissues. The latter are predominantly located in DCVs with a lesser contribution from the Golgi apparatus from which they arise (24). Ultrastructural observations in the current investigation were limited to two neuronal processes in the pharyngeal nerve ring that were presumed to originate from the ALA cell body and the uv-1 neuroendocrine cells in the region of the vulva. Both cell types contained electron-dense spherical objects of 50 nm diameter that were strongly immunoreactive with GFP antibodies consistent with their identification as DCVs. The sorting of phogrin to DCVs in mammalian neuroendocrine cells appears to be driven by conformational structural components in the lumenal domain (58) with a secondary contribution from the juxtamembrane region of the cytosolic domain (Wasmeier & Hutton, unpublished observations). ICA512, phogrin and IDA-1 possess an evolutionarily conserved YxxF internalization motif within the latter region (20), suggesting that these molecules are actively endocytosed for either recycling postexocytosis or degradation. Although the molecules may pass through elements of the endocytic pathway, there appears little contribution to the steady state distribution of the molecules. It is conceivable that some of the 554
observed fluorescent punctae in the nematode neuron could represent recycling components rather than DCVs destined for exocytosis. Such components, as in the case of mobile DCVs, are smaller than 250 nm in diameter based on epifluorescence microscopy and may be identifiable in future studies using specific endosomal markers.
Dynamics of IDA-1::GFP-tagged vesicles In vivo time-lapse microscopy of IDA-1::GFP transgenic animals showed complex trafficking behaviors. Saltations (Figure 6C), reversals (Figure 6G) and transport velocities in the order of 1 mm/s (Figure 7) are characteristic of vesicular MT-based fast axonal transport and have been observed directly by light microscopy of neuronal organelles in both vertebrate and invertebrate model systems (53,54 and references therein), as were simultaneous and parallel movements (Figures 6E,F) and the disruption of movements of different particles at the same location (Figure 6H). It was not obvious that IDA-1::GFP movements in either anterograde or retrograde directions had any particular ‘destination’ corresponding for example to transfer of vesicles to an exocytic site or return of DCV proteins to the trans-Golgi network for recycling. In light of the frequent reversal of direction, the movements were more consistent with a model of motor-assisted diffusion in which vesicles shuttle randomly in both directions along neuronal processes until they reach a target site (59).
Neuronal polarity of the molecular transport machinery Neuronal function requires specialization and polarity of processes that dictate the efficient sorting and differential segregation of proteins, organelles and mRNAs. The polarity of MTs and their motors provides one important mechanism in this context, as kinesins and dyneins are polarized in their activity and move unidirectionally along MTs (8). In the limited number of both vertebrate and invertebrate neuronal cell types that have been analyzed, MT orientation itself was polarized with plus-ends distal to the cell body in axons (60,61 and references therein), while in dendrites of cultured rat hippocampal neurons, MT polarity orientation was mixed and only uniform near the dendrite tip (also with plus-ends distal) (60,62). As a result of uniform MT polarity orientation within axons, motors can move actively only in one direction within the axon and for movement in the opposite direction would have to be returned as cargo by another carrier. The polarity of MTs in C. elegans neuronal processes has yet to be determined; however, the observed differences between anterograde and retrograde vesicle velocities within individual neuronal processes (e.g. Figure 7A) are most easily explicable if MTs are oriented unidirectionally within a process. A uniform MT orientation with plus-ends distal is also consistent with the velocity phenotype in mutants defective in the MT plus-end directed motor protein UNC-104 (Figure 8B). Traffic 2004; 5: 544–559
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The observation of two distinct classes of velocity profiles discriminated by the presence or absence of retrograde transport velocities >1.5 mm/s (Figure 7) implies that different neurons utilize common sets of neuronal motor proteins that are differentially sorted into axonal and dendritic processes. The ALA lateral processes and PHC posterior processes are examples of the two different velocity profiles and are likely representative of axons and dendrites, respectively.
Velocities of IDA-1::GFP transport Its transparency and thin neuronal processes make C. elegans well suited for in vivo observation and quantitative analysis of neuronal transport events. In C. elegans, neuronal transport velocities have been measured for several components of the amphid sensilla. Intraflagellar transport along cilia in the specialized sensory endings of amphid neurons was reported for a number of GFP-tagged proteins to occur at 0.7 mm/s anterogradely and 1.1 mm/s retrogradely (29,30,63). Within amphid dendrites a GFP fusion protein of the odorant receptor ODR-10 was transported at 1.42 0.09 mm/s in anterograde and 0.71 0.04 mm/s in retrograde direction (28), while OSM1::GFP and OSM-6::GFP proteins moved at a similar speed as the kinesin II subunit KAP::GFP (0.67 0.09 mm/s anterogradely and 1.07 0.10 mm/s retrogradely in the case of the latter) (30). These observations all have in common single unimodal and well-defined velocities, suggesting that each of these cargoes are transported by a single motor for each direction. Such a notion is further supported by the observation that retrograde transport in cilia was absent in che-3 dynein mutants (30). In contrast, the velocity distributions of IDA-1::GFP transport appear novel and remarkably complex with regard to their multiple, distinct velocity peaks. The similarity of velocity profiles in different processes (Figures 7 and 8) argues that the multiple peaks reflect a shared biological phenomenon. Each peak of the characteristic IDA-1::GFP transport velocity profiles (Figure 7) could reflect the intrinsic speed of a particular motor protein. However, the number of different motor proteins could be smaller than the number of peaks due to regulation of motor processivity by protein phosphorylation, for instance (64). Also, intermediate and faster velocities could be the result of cooperation between multiple motors acting on the same vesicle, and slower velocities could be caused by a ‘tug-of-war’ between motors of opposing directions (65,66). Conversely, motors with similar velocities present in the same neuronal process would be represented by the same peak. Supported by the unc-104 data (Figure 8), it can be concluded that IDA-1::GFP anterograde transport in at least some neuronal processes with axonal characteristics is mediated by two or more different molecular motors. This implies that while some proteins and organelles are transported by
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a single specific motor (30), others are promiscuous and utilize multiple different motors.
In vivo velocity of kinesin UNC-104 In vitro assays with experimentally defined concentrations of motor proteins, ATP and other cofactors have provided detailed insights into the molecular mechanisms underlying processive motor movement (11). In vivo studies of motor proteins supplement our knowledge of molecular motors in regard to cargo specificity and their activity in a physiological context. In unc-104 mutant animals, IDA1::GFP accumulated in cell bodies (Figure 5), and anterograde DCV transport was impaired correspondingly, with a striking loss of the fastest anterograde velocities (Figure 8). The velocity defect was seen specifically in ALA lateral processes but not PHC posterior processes, underscoring the difference in the transport machinery of these neurites. The mutant allele used here, e1265, carries a mutation in the cargo-binding pleckstrin homology domain and has severely reduced UNC-104 protein levels (S. P. Koushika and M. L. Nonet, personal communication). Our results imply that DCVs are a cargo with multiple different motors in C. elegans neuronal processes, with UNC-104 responsible for anterograde axonal (but not dendritic) transport at in vivo rates of 1.6–2.7 mm/s. These velocities are within the reported range of velocities measured for an UNC-104::GFP fusion protein (31), although the average velocity in this study was determined as 1.02 0.53 mm/s. In vitro velocities measured in MT gliding assays with forced UNC-104 dimers were very similar to the in vivo UNC-104 velocities reported here (1.84 0.50 mm/s for single units and 2.72 0.17 mm/s for multiple units of the U403-Kstalk2-GFP chimera dimer) (67). Importantly, this study by Tomishige et al. (67) showed a dependence of UNC-104 activity on the degree of dimerization with higher velocities for multiple UNC-104 dimers compared to single motor units, providing a possible explanation for the relatively broad range of IDA-1::GFP anterograde transport velocities affected in ALA lateral processes of unc-104 mutants. The generation of IDA-1::GFP transgenic nematodes presents an opportunity to explore many aspects of neurosecretory behavior at the cellular and subcellular level. The use of the nematode for studies of peptidergic secretion has been less extensive than its use for studies on synaptic neurotransmission of classical low molecular weight neurotransmitters. Yet C. elegans uses peptide secretion to regulate much of its physiologic and metabolic responses to the environment, including molecules such as insulin, which in higher vertebrates perform a specific endocrine function. The observation that mutants in the kinesin family of molecular motors generate a macroscopic and scoreable phenotype based on the distribution of IDA1::GFP in cell body vs. distal regions of the neuron paves the way to performing an unbiased genetic screen. Genes that are essential for the biogenesis of the DCV, its sorting
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and transport, and the molecular cell biology of polypeptide neurotransmission may be identified by such a screen.
Materials and Methods Generation of anti-IDA-1 antibodies A plasmid construct encoding a glutathione-S-transferaseIDA-1-C-terminus fusion protein was generated by inserting a polymerase chain reaction (PCR)-product (forward primer 50 -CGCTAGGATCCGTCGTCATTACAAGGA-30 , reverse primer 50 -GCATCTCGAGGGGAACAACGATTTTT-30 from cDNA yk27f7 (GenBank accession number AJ245560) (20), coding for the C-terminal domain of IDA-1 (aa 427–767) into vector pGEX-5X-3 (Amersham Pharmacia Biotech, Piscataway, NJ) using BamHI and XhoI restriction sites. The soluble 65 kDa fusion protein was produced in Escherichia coli (XL1 Blue), affinity-purified on glutathione Sepharose (Amersham Pharmacia Biotech) and mixed with Freund’s complete adjuvant to immunize NZ white rabbits. Anti-IDA-1 antibodies were purified using the fusion protein coupled to CNBr-activated Sepharose (Amersham Pharmacia Biotech) as an affinity support and concentrated by ultrafiltration (Centriplus; Amicon, Beverly, MA). Immunostaining N2 nematodes (mixed stage) were fixed in modified Bouin’s fixative (35.2% saturated picric acid, 11.8% formalin, 49.4% methanol, 2.4% acetic acid, 1.2% 2-mercaptoethanol by volume) (41) for 30 min, freeze-thawed using liquid nitrogen, and postfixed for 30 min. They were then washed four times with BT buffer (20 mM H3BO3 pH 9.3, 0.5% Triton X-100) containing 2% 2-mercaptoethanol over 3 h, once with BT alone for 30 min, then twice with AbA buffer (phosphate-buffered saline (PBS), 1% bovine serum albumin, 0.1% Triton X-100, 0.05% NaN3) and equilibrated in AbA for 30 min. The samples were incubated overnight with affinity-purified anti-IDA-1 antibodies (1 : 100 in AbA), washed four times in AbA over 4 h, incubated overnight in Cy2-conjugated donkey antirabbit IgG (1 : 100 in AbA) (Jackson ImmunoResearch Laboratories, West Grove, PA), and washed three times in AbA and then once in PBS overnight and mounted in moviol. Affinity-purified antisera from three different rabbits gave similar results. Immuno-electron microscopy Several hundred late L4 BL5752 worms were scraped from culture dishes together with E. coli (which serves as a cryoprotectant) and placed in brass freezing planchettes (type A from Ted Pella, Redding, CA), then fastfrozen with liquid nitrogen in a BAL-TEC HPM 010 high pressure freezer (Technotrade International, Manchester, NH). The planchettes were mechanically separated under liquid nitrogen and placed into cryotubes (Nalge Nunc, Naperville, IL) containing a frozen solution of 0.25% glutaraldehyde (EMS, Ft. Washington, PA) with 0.01% uranyl acetate (EMS) in acetone and warmed to 90 °C to allow 556
freeze-substitution using a Leica EM AFS freeze substitution device (Leica Microsystems, Vienna, Austria) over 96 h. Samples were then warmed to 50 °C over a period of 40 h (0.1 °C/ h) at which point they were transferred into porous (78 mm) microcapsules (EMS) to limit the number of worms lost during infiltration. The material was then washed three times with cold anhydrous acetone and infiltrated with dilutions of 30% Lowicryl HM20 (EMS) in acetone at 50 °C for 24 h, 60% HM20 in acetone for 24 h, then 3–4 changes of 100% HM20 over a 3-day period. Following infiltration, the samples were transferred to flat-embedding moulds (Sigma, St. Louis, MO) and polymerized at 50 °C using UV light in the AFS unit. The flat-embedded worms were then warmed to room temperature, excised, and oriented onto an Epon block for sectioning (68). Sections of 100 nm were mounted on formvar-coated nickel slot grids and stained with anti-GFP affinity-purified polyclonal rabbit antibody (69) (1 : 150) and then with goat antirabbit IgG secondary antibody labeled with 10 nm colloidal gold (1 : 20) (Ted Pella). The grids were postfixed in 1.0% glutaraldehyde in PBS, stained with uranyl acetate and lead citrate and examined in a Philips CM-10 transmission electron microscope operating at 80 kV. Cloning of the reporter construct The transcriptional reporter construct pida-1::GFP 2.4 (20) was digested with PstI and KpnI and religated (fill-in). A PCR product from ida-1 cDNA yk27f7 (T3 promoter forward primer 50 -ATTAACCCTCACTAAAGGGA-30 ; reverse primer 50 20 -TTATGCATGCGATTGACTTTAGAAG-30 ) was digested with SphI and inserted into the SphI-cut vector. A BamHI-NheI ida-1 genomic fragment from cosmid B0244 was generated to replace the corresponding BamHI-NheI cDNA fragment, thereby generating the construct pida-1::IDA-1::GFP, which contains a 2443 bp ida-1 promoter upstream of an ida-1 coding region that incorporates introns 3–9 followed at the 30 -end with in-frame GFP coding sequence and terminating with the unc-54 30 untranslated region. C. elegans strains Wild-type C. elegans (variety Bristol, N2) and the following recessive mutations were used: aex-3(sa5), cab-1(tg46), egl-3(nr2090), rab-3(js49), rme-1(b1045), rpm-1(ju41) V, sad-1(ky289), snb-1(md247), snt-1(n2665), syd-2(ju37), unc-11(e47), unc-13(e51), unc-14(e57), unc-31(e928), unc51(e369), unc-64(e246), unc-104(e1265), unc-104(rh43). Transgenic nematodes were generated by gonadal injection of N2 hermaphrodites with the DNA plasmid construct pida-1::IDA-1::GFP (100 ng/mL). Stable extrachromosomal strains expressing IDA-1::GFP were g-irradiated (3800 rad, Cs source) for integration of the transgene and progeny cloned to select strains segregating 100% IDA-1::GFPexpressing worms. Two independent lines were outcrossed for two generations with male N2 worms to generate strains BL5750 (inIs181 IV) and BL5751 (inIs182 I), Traffic 2004; 5: 544–559
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and the double-transgenic strain BL5752 was generated by intercrossing strains BL5750 and BL5751 and selecting for high IDA-1::GFP expression levels. Transgenic mutant strains were generated by appropriate genetic crosses between the mutant strain and BL5752, animals were identified by their visible phenotype or alternatively by sequencing to verify the mutant genotype (rab-3, rpm-1, sad-1, snb-1, syd-2). Western blotting N2, BL5752, and BL5778 (egl-3(nr2090)); IDA-1::GFP) transgenic worms were raised at 20 °C, boiled in SDSPAGE loading buffer, and equivalent amounts of protein from each strain were separated by SDS-PAGE (7.5% acrylamide). Proteins transferred to nitrocellulose membranes were probed with an affinity-purified polyclonal rabbit anti-GFP antibody (69) (1 : 500 dilution) followed by protein A-HRP (1 : 6700): and chemiluminescence reaction (Western Lightning, Perkin Elmer). C. elegans microscopy For in vivo microscopy, young adult hermaphrodites were immobilized with 10 mM levamisole (Sigma-Aldrich) in M9 buffer and mounted under a coverslip on a 2% agarose pad on a microscope slide. Alternatively, worms were preincubated for 1 h in 100 mM nocodazole, 0.1% DMSO in M9 buffer or mounted directly in 5 mM NaN3 without levamisole. DMSO did not alter vesicle velocity profiles in control animals and all drug effects appeared fully reversible. Images were recorded at 20 1 °C with a SensiCam CCD-camera (Cooke, Auburn Hills, MI) coupled to a G4 Macintosh computer (Apple, Cupertino, CA) running Slidebook software (Intelligent Imaging Innovations, Denver, CO) using a Zeiss Axiovert 100 inverted microscope (Carl Zeiss, Thornwood, NY) equipped with a 63 1.4 n. a. oil objective and a Sedat Quad Filter Set (Chroma, Brattleboro, VT). Fast time-lapse images were acquired at 4– 6 Hz (depending on image size) using 100 ms exposures. Slow time-lapse images of ALA neurons were performed at 1-h intervals using a series of 500 ms exposure and 8 neutral density filter attenuation of the exciting light. A collage was assembled from 25–40 images using Photoshop software (Adobe Systems, San Jose, CA). For quantitation of IDA-1::GFP fluorescence, eight or more animals each of mutant and wild-type transgenics were imaged in the same session to avoid differences in illumination intensity. Data analysis Kymographs (52) were generated using Slidebook’s ThreeView tool after rotation of the image stack to align the neuronal process vertically. Vertical single line-scans through the center of each process were plotted sequentially for every frame in the time series. Excluding animals not fully immobilized by levamisole, distance and velocity measurements for uninterrupted IDA-1::GFP movements at constant velocity spanning at least five timepoints were recorded in Excel software (Microsoft, Redmond, WA) for further analysis. Each pixel of the CCD-camera chip repreTraffic 2004; 5: 544–559
sents an image area of 106 nm 106 nm, causing a systematic error for single velocity measurements of up to 10% (depending on the duration of the transport event). Igor (WaveMetrics, Lake Oswego, OR) was used to generate histograms and for curve fitting. Best fits were obtained with the multifit XOP integer. SPSS software (SPSS, Chicago, IL) was used to calculate the KolmogorovSmirnov test for normal distribution. About 80 data points seemed sufficient to generate a representative histogram whose overall shape and distinct velocity clusters would not be significantly altered by inclusion of additional data points. Our data analysis differed somewhat from the approaches used in most previous studies. Historically, velocities have mostly been measured as mean velocities, calculated as total distance traveled divided by the observation time. Velocities determined this way were well suited for comparison with the transport rates known from the bulk flow of proteins measured over time frames of hours and days as they reflected the average activity of the neuronal transport machinery regardless of intermittent velocity changes; however, they did not directly reflect the ‘in situ’ activity of individual working motor proteins. The calculation of ‘instantaneous velocities’ from organelle displacements frame-by-frame did include such measures of motor velocity, but measurements of such very short distances are much less accurate due to the limited spatial resolution. Here we restricted velocity measurements to movements occurring at apparently constant velocity to reflect motor velocities during processive movement but of sufficient duration to minimize distance measurement errors. Net fluorescence intensity (Figure 5) was calculated as the difference between the integrated fluorescence intensity from an area of interest and that of an adjacent background area of identical size in Slidebook; statistical significance was determined with the Student’s t-test using Excel software. Images were processed for publication in Slidebook, Photoshop and Illustrator (Adobe Systems). Quicktime videos were generated in Slidebook.
Acknowledgments We thank Yuji Kohara for the ida-1 cDNA, the Washington University Genome Sequencing Center (St. Louis, MO) for the ida-1-containing cosmid B0244, Theresa Stiernagle and the NIH-funded Caenorhabditis Genetics Center and Tija Jacob and Josh Kaplan for mutant strains, Mary Morphew for anti-GFP antibodies and help with C. elegans immuno-electron microscopy, Sandhya Koushika and Mike Nonet for communication of unpublished results, Nelson Huang for assistance with the nocodazole experiment, Lars Stene for help with the statistical analysis, Richard McIntosh for advice on ultrastructural analysis, Steve Fadul for help with confocal microscopy, Colin Monks (Intelligent Imaging Innovations, Denver, CO) for help with the image analysis, Zeynep Altun for advice on C. elegans anatomy, and Tom Blumenthal for discussions and support. Funding was provided by the National Institutes of Health (R21 DK60861) and the Children’s Diabetes
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Zahn et al. Foundation. Microscopy work at the Light Microscopy Core Facility at UCHSC is supported by the NIH grant S10 RR14648 to W. J. Betz and electron microscopy was supported by the NIH Biotechnology Resources grant RR00592 to J. R. McIntosh. Sequencing was provided by the Molecular Biology Core Services supported by the NIH DERC grant P30-DK57516.
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