Mutations in Caenorhabditis elegans cytoplasmic dynein components reveal specificity of neuronal retrograde cargo

Abstract

We describe Caenorhabditis elegans dynein complex mutants, which misaccumulate synaptic proteins at the ends of neuronal processes. Ultrastructural analysis revealed irregularly sized vesicles that likely represent accumulation of cargo. We propose that synaptobrevin, synaptotagmin, and UNC-104 are specific cargoes of the dynein complex. Many cargoes link to dynein via interactions between dynactin and vesicle-associated spectrin. However, loss of spectrin results in only mild and occasional defects in synaptobrevin localization. Thus, the dynein-dynactin complex shows neuronal cargo selectivity without spectrin being a critical component of cargo binding. We observed parallels to progressive motor neuron disease symptoms in these animals. With age, neuronal misaccumulations increase in size and frequency; locomotion becomes progressively slower; and life span is shortened. These mutants provide a model to assess whether defects in transport of specific cargo mediate neuronal dysfunction.

Figure 1.

Neuronal architecture of misaccumulation mutants. Top, Schematic diagram of worms expressing synaptobrevin::GFP in mechanosensory neurons. ALMs are a pair of lateral mechanosensory cells that extend neuronal processes into the nose and nerve ring. a-c, jsIs37(a), dhc-1(js319); jsIs37(b), anddli-1(js351); jsIs37(c) noses; bhas one misaccumulation, whereas the neuronal process in c has multiple misaccumulations. The arrowhead in a shows the main ALM neuron process, and arrows in b and c show misaccumulations of SNB-1::GFP near the tip of ALM processes. d-f, jsIs37, dhc-1(js121); jsIs37, and dli-1(js351); jsIs37 nerve ring area. Mutants have hyperaccumulations of synaptobrevin expression indicated by the arrows. g-i, jsIs37, dhc-1(js319); jsIs37, and dli-1(js351); jsIs37 patches in the ventral cord, which appear grossly similar. j-l, jsIs37, dhc-1(js121); jsIs37, and dli-1(js351); jsIs37 immunostaining for syntaxin, which marks all neuronal processes. Seen here is an image of the dorsal cord and neuronal commissures; the mutants appear similar to wild type. m-o, jsIs37, dhc-1(js121); jsIs37, and dli-1(js351); jsIs37 immunostained for RAB-3 (green) and Rim (red) from the ventral cord. Mutant animals are indistinguishable from wild type. Scale bars: 10 μm; o, 5 μm.

Figure 2.

Ultrastructure of dhc-1 and dli-1. a-c, Ventral cord neuromuscular junction electron micrographs of 1- to 2-d-old adult animals: wild type (a), dhc-1(js121); jsIs37 (b), and dli-1(js351); jsIs37 (c). The arrow points to the active zone, and the profile shows the presence of synaptic vesicles. d-g, Electron micrographs of ALM neuron process showing the large-diameter microtubules in wild type (d) and dhc-1(js121); jsIs37 (e) 1- to 2-d-old adults. f, g, Collection of vesicles (arrows) in dhc-1(js121); jsIs37 ALM process in 1- to 2-d-old adult animals. Scale bar, 200 nm.

Figure 3.

Misaccumulations in the nose are dependent on a kinesin family motor and microtubules. a, The arrow points to the ALM process in jsIs37. b, The arrow points to a misaccumulation at the tip of the ALM process in dhc-1(js121); jsIs37. c-e, Phenotype of worms grown in 0.8 mm colchicine.c,jsIs37, no signal in nose or nerve ring. d, dhc-1(js121); jsIs37, some signal in the nervering (arrowhead) but no misaccumulation of SNB-1::GFP in the nose. e, dli-1(js351);jsIs37, some signal in the nerve ring (arrowhead) but SNB-1::GFP misaccumulation in the nose is absent. f-h, unc-104 kinesin-like protein double mutants. f, unc-104(e1265); jsIs37 animals have no signal in the nose. g, dhc-1(js121); unc-104(e1265); jsIs37. Some animals have misaccumulations (arrow), but they are significantly smaller (compare with b). h, unc-104(e1265); dli-1(js351); jsIs37. Misaccumulations are absent, although some GFP is present in the nerve ring (arrowhead). Scale bar, 10 μm.

Figure 4.

Dynein complex mutants misaccumulate the anterograde kinesin family motor UNC-104 and native synaptobrevin. a-d, GFP (first image of set) and UNC-104 (second image of set) Ab staining. a, b, dhc-1(js319); jsIs37 SNB-1::GFP misaccumulations (arrow) in the nose also contain the UNC-104 protein. The arrowhead in b points to UNC-104 protein in a SAB neuron. c, d, dli-1(js351); jsIs37 SNB-1::GFP misaccumulations (arrow) in the nose also contain UNC-104 protein. e, f, Dynein heavy chain immunostaining. e, Nerve ring staining in wild type showing that the heavy chain is enriched in neurons. f, Immunoreactivity in unc-104(e1265) animals still shows DHC-1 protein enriched in the nerve ring. g, Double labeling for synaptotagmin (green) and synaptobrevin (red) staining in dhc-1(js319) animals lacking jsIs37 showing a misaccumulation containing both proteins (arrow). Scale bars, 10 μm.

Figure 5.

Localization of synaptic proteins in dynein mutants. In all images, arrows point to misaccumulations at the tip of ALM neurons. Arrowheads point to synaptic vesicle protein in an SAB neuron. a-f, Double labeling of GFP (green, first image of set) and SNT-1 (red, second image of set) immunoreactivity. a-c, dhc-1(js121); jsIs37. d-f, dli-1(js351); jsIs37. SNB-1::GFP misaccumulations in both genotypes contain SNT-1 immunoreactivity. g-i, Double labeling of GFP (green, first image of set) and RAB-3 (red, second image of set) immunoreactivity of dhc-1(js121); jsIs37. j-l, GFP (green, first image of set) and transmembrane protein SNG-1 (red, second image of set) immunoreactivity of dhc-1(js121); jsIs37. SNB-1::GFP misaccumulations do not contain either RAB-3 or SNG-1 immunoreactivity. m-r, Double labeling for GFP (green, first image of set) and UNC-64 syntaxin (red, second image of set) immunoreactivity. m-o, dhc-1(js121); jsIs37 SNB-1::GFP containing misaccumulations also contain syntaxin. p-r, dli-1(js351); jsIs37. Two SNB-1::GFP misaccumulations are present in the nose, one of which contains syntaxin (arrow) and one of which does not (arrowhead). In general, ∼40-50% of SNB-1::GFP misaccumulations contain syntaxin. The buccal cavity of the animal often shows nonspecific reactivity with many antibodies; this is seen in b, c, e, f, i, n, o, q, and r. Scale bar, 10 μm.

Figure 6.

Misaccumulations of protein in dnc-1(or404ts) p150^Glued mutant and in overexpression of dnc-2 dynamitin in mechanosensory neurons. All arrows point to misaccumulations at the tip of the ALM process in the nose. a-f, Double labeling for GFP (green, first image of set) and SNT-1 (red, second image of set) immunoreactivity. a-c, dnc-1(or404ts). d-f, dnc-2 overexpression. g-l, Double labeling of GFP (green, first image of set) and UNC-104 (red, second image of set) immunoreactivity. g-i, dnc-1(or404ts). j-l, dnc-2 overexpression. m-r, Double labeling for GFP (green, first image of set) and UNC-64 syntaxin (red, second image of set) immunoreactivity. m-o, dnc-1(or404ts). p-r, dnc-2 overexpression. SNB-1::GFP misaccumulations contain SNT-1, UNC-104, and UNC-64 immunoreactivities. The buccal cavity of the animal often shows nonspecific reactivity with many antibodies; this is seen in f, h, i, k, l, n, o, q, and r. Scale bar, 10 μm.

Figure 7.

Role of spectrin in localization of SNB-1::GFP in mechanosensory neurons. a, Nose of dhc-1(js121); jsIs37, where the arrow shows an accumulation. b, unc-70(e524); jsIs37, which does not have any misaccumulation.c, unc-70(s1502); jsIs40 with an accumulation (arrow). d, sma-1(ru 18); jsIs37 animal that has a misaccumulation (arrow). No hyperaccumulations are seen in the nerve ring regions of Unc-70 animals (a, arrowhead; data not shown). Scale bar, 10 μm.

Figure 8.

Age dependent effects of the mutants. A, Size of accumulations of 1- and 5-d-old adult dhc-1(js121); jsIs37 and dli-1(js351); jsIs37 animals. All abnormal accumulations in a single neuronal process were measured for a data point. See Figure 1, b and c, for how numbers of misaccumulations can vary in a single neuronal process. The increase in size of misaccumulations is nearly 2.5 times as the animal ages (n = 20). The 1 and 5 d values are significantly different for both genotypes (p < 5 × 10^-6). Data are presented as area ± SEM. B, Change in movement with age. Velocity of movement after a fixed stimulation of wild-type jsIs37, dhc-1(js121); jsIs37, and dli-1(js351); jsIs37 adult animals of 1 d (n = 21) and 5 d (n ≥ 17). Velocity measurements of both mutants are significantly different from those of wild-type animals of corresponding age (p < 5 × 10^-4). Data are represented as velocity ± SD. C, Life span curves. Animals were collected as late L4 larvae (0 d) and followed every other day until they died. Wild-type jsIs37, dhc-1(js121); jsIs37, and dli-1(js351); jsIs37 animals have n = 35, 26, and 23, respectively.

Figure 9.

Neuromuscular junctions as observed by SNB-1::GFP expression in VD and DD motor neurons. a-d, One-day-old adult animals of wild-type juIs1 (a, c) and dhc-1(js121); juIs1 (b, d). e-j, Five-day-old adult animals of wild-type juIs1 (e, g, i) and dhc-1(js121); juIs1 (f, h, j). Arrowheads point to clumps of SNB-1::GFP, gaps in SNB-1::GFP puncta and other irregularities in SNB-1::GFP localization in mutant animals. Scale bar, 5 μm.