Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170

Abstract

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

Figure 1

Homology plots of chicken versus human and fly dynamitin sequences. Plots were drawn to highlight the regions of highest homology between species. Chicken, GenBank accession number AF200744; human, accession number AAC50423; fly, for AA 1–184, EST 81725; for AA 297–380, EST AA802366, for AA 1–34 and 137–380, genomic clone AC007471.

Figure 2

Effect of HA-dynamitin overexpression on the steady-state localization of early endosomes, late endosomes, and TGN. Cells were double labeled for dynamitin (HA; a, c, e, and g), early endosomes (EEA1, b; Tfn-R, f), late endosomes (LBPA; d), and TGN (MPR, mannose-6-phosphate receptor; h). Cells overexpressing HA-dynamitin are marked with asterisks. HeLa cells in a–d were fixed with 3% paraformaldehyde, and cells in e–h were fixed with MeOH. Bar, 20 μm.

Figure 3

Dynamitin N terminus disrupts Golgi organization but not dynactin structure. (A) Cos7 cells transfected with the highly conserved dynamitin N terminus (AA 1–87) were stained for dynamitin (pAb dynamitin; left) and the Golgi complex (giantin; right). (B) Lysates of Cos7 cells transfected with β-gal, dynamitin N terminus, or HA-dynamitin were analyzed by velocity sedimentation (Echeverri et al., 1996). Transfection efficiency was ≥50%. Gradient fractions were immunoblotted with antibodies to p150^Glued, p62, and Arp1. Sedimentation standards are indicated.

Figure 4

Tfn trafficking in control and dynamitin-overexpressing cells. (A) HeLa cells were labeled with FITC-Tfn for 1 h and then fixed (paraformaldehyde followed by MeOH) and stained for the late endosome marker LBPA (left). Inset, boxed area at 2× magnification. Cells overexpressing HA-dynamitin are marked with asterisks. (B) Qualitative analysis of uptake. Transfected Cos7 cells were labeled with Cy3-Tfn for 20 min and then fixed. Dynamitin overexpressers (i.e., cells with disrupted Golgi complexes) and control cells were scored as bright or dim (n = 180 cells total). (C) Quantitative analysis of Tfn uptake and recycling. HeLa cells were fixed after increasing times of labeling (0–60 min) or after 1 h of labeling and 0–60 min of chase. The fluorescence intensities of ≥15 control (solid lines) or dynamitin-overexpressing (dotted lines) cells were measured, and mean values are plotted. Bar in A, 20 μm.

Figure 5

Analysis of endocytic trafficking in control and dynamitin-overexpressing cells. (A) HeLa cells were labeled with Cy3-α₂-M for 10 min and then chased for 5 min and fixed with paraformaldehyde. The same cells were then stained for early endosomes (EEA1; left) and overexpressed dynamitin (right); α₂-M staining is shown in the middle. (B) As in A, except the cells were chased for 1 h after the α₂-M pulse to allow the probe to traffic to late endosomes. After fixation, the cells were stained for EEA1 (left) and LBPA (right); α₂-M staining is shown in the middle. For A and B, the small panels to the right are 3× magnification views of the boxed areas; cells overexpressing HA-dynamitin are marked with asterisks. Arrows in A indicate early endosomes that also stain for α₂-M. (C) Qualitative analysis of α₂-M uptake and accumulation. Transfected Cos7 cells were labeled for 20 min and then fixed immediately (left panel) or chased for 90 min and then fixed (right panel). The fluorescence intensity (bright, dim, or no label) of ≥150 cells was assessed for each condition. (D) Subcellular distribution of endosomes loaded with Tfn or α₂-M. Transfected HeLa cells were loaded with fluorescent tracers, fixed immediately, and then scored for endosome location. Central, random, and peripheral indicate the site of the most predominant staining. Similar results were obtained in Cos7 cells (our unpublished results). (E) Analysis of endosome pH in transfected, live Cos7 cells. pH was measured by fluorescence ratio imaging (Kim et al., 1996). Data were acquired from at least two different coverslips in two independent experiments. Dynamitin overexpressers were identified by staining with anti-HA (A, B, and D) or pAb dynamitin (C) or by Golgi morphology (E).

Figure 6

Effect of dynamitin overexpression on virus infection and protein export. (A) Vero cells transfected with dynamitin (top panels) or GFP (bottom panels) were infected with ts-O45 VSV for 1 h at 17–20°C and maintained at 39.5°C to retain VSV-G protein in the ER. After 3 h, cells were fixed and stained for dynamitin (HA; top left) or GFP (bottom left) and VSV-G protein (right top and bottom, mAb P5D4 and polyclonal serum). Cells overexpressing HA-dynamitin are marked with asterisks. Bar, 20 μm. (B) Quantitation of infection. Vero cells were microinjected (gray bars) with HA-dynamitin or Sar1p (a yeast rab) or transfected (black bars) with HA-dynamitin, GFP, or β-gal and infected for 1 h at room temperature (RT; 17–20°C). Control cells that received no DNA (white bar) were infected in parallel. Cells were stained, and the percent expressing both the exogenous protein and ts-O45-G protein was determined. At least 220 dynamitin-overexpressing cells, in four independent experiments, were counted for each condition. Sar1p overexpression was documented on two separate coverslips in a single experiment. The GFP bar shows data from GFP and β-gal transfections. In the remaining five black bars, cells were infected for 1 h at RT, 1.5 h at 20°C, 2 h at 20°C, 1 h at 37°C, or 2 h at 37°C.

Figure 7

Effects of dynamitin overexpression on colocalization of CLIP-170 and dynactin at microtubule ends. (a and b) Control cells costained for CLIP-170 and dynactin (Arp1). (c and d) High-magnification view of two cells overexpressing dynamitin at a low level showing staining of peripheral microtubule ends. (e and f) High-level dynamitin overexpression (asterisks in e marks overexpressing cells). HeLa cells were stained for CLIP-170 (pAb 55, a; mAbs 2D6 and 4D3, c and e), dynactin (Arp1 mAb 45A, b) or dynamitin (pAb anti-HA, d and f). All were fixed with MeOH. Bars, 20 μm. Magnification in insets a1–b3, 4×.

Figure 8

Effects of p150^Glued and CLIP-170 overexpression on CLIP-170 and dynactin localization. (a and b) p150^Glued overexpression. (c–f) Effects of wt CLIP-170 overexpression on p150^Glued (c and d) and Arp1 (e and f) staining. (g and h) Overexpression of CLIP-170ΔT (lacking the C-terminal domain that contains the metal-binding motif). HeLa cells were stained for CLIP-170 (pAb 55, a and e; 2D6 and 4D3, c; anti-myc mAb 9E10, g) or dynactin (p150^Glued: mAb 150.1, b; pAb 150^Glued, d and h; Arp1: mAb 45A, f). All were fixed with MeOH. Bars, 20 μm. Inset magnifications, 2×.

Figure 9

Effects of CLIP-170 overexpression on Golgi complex, late endosome, and lysosome distributions. HeLa cells were fixed with MeOH and stained with Abs to (a and c) CLIP-170 (pAb 55), (b) galactosyl transferase, and (d) CD63 (a LAMP family member; mAb 1B5). Bar, 20 μm.