Dynein binds and stimulates axonal motility of the endosome adaptor and NEEP21 family member, calcyon

Abstract

The neuron-enriched, endosomal protein Calcyon (Caly) regulates endocytosis and vesicle sorting, and is important for synaptic plasticity and brain development. In the current investigation of Caly interacting proteins in brain, the microtubule retrograde motor subunit, cytoplasmic dynein 1 heavy chain (DYNC1H), and microtubule structural proteins, α and β tubulin, were identified as Caly associated proteins by MALDI-ToF/ToF. Direct interaction of the Caly-C terminus with dynein and tubulin was further confirmed in in vitro studies. In Cos-7 cells, mCherry-Caly moved along the microtubule network in organelles largely labeled by the late endosome marker Rab7. Expression of the dynein inhibitor CC1, produced striking alterations in Caly distribution, consistent with retrograde motors playing a prominent role in Caly localization and movement. In axons of cultured adult rat sensory neurons, Caly-positive organelles co-localized with dynein intermediate chain (DYNC1I1-isoform IC-1B) and the dynein regulator, lissencephaly 1 (LIS1), both of which co-precipitated from brain with the Caly C-terminus. Manipulation of dynein function in axons altered the motile properties of Caly indicating that Caly vesicles utilize the retrograde motor. Altogether, the current evidence for association with dynein motors raises the possibility that the endocytic and cargo sorting functions of Caly in neurons could be regulated by interaction with the microtubule transport system.

Keywords: AP-1; AP-2; AP-3; Adaptor protein; Brain; Clathrin; Endocytosis; Lysosome; Schizophrenia; Tubulin.

Figure 1. Association of brain microtubule proteins with Caly

A. SYPRO Ruby stained 1-D SDS gel of proteins eluted from resin-bound GST (left lane) or GST-Caly-C (middle lane) following incubation with homogenates of mouse forebrain (right lane). Numbered arrows point to two bands picked for trypsin digestion and subjected to MALDI-TOF MS that corresponded to microtubule related proteins. Asterisks show the position of the GST and GST-Caly-C bands in the GST and GST-Caly-C pull-down lanes, respectively. β tubulin (B) or DIC (C) antibody probing of immunoblots of proteins eluted from GST or GST-Caly following incubation with highly purified α/β tubulin or dynein complex, respectively. D. SYPRO Ruby stained PVDF membrane showing the subunits of purified dynein complex, including heavy chain (HC), intermediate chain (IC), and light intermediate chains (LICs). Purified S-protein tagged Caly-93-217 was incubated overnight with the membrane, and binding visualized with S-HRP. A single band corresponding to IC was detected by ECL.

Figure 2. Localization and movement of Caly along the microtubule network

mCh-Caly (A,D) and GFP-tubulin (B,E) co-transfected Cos-7 cell. Region in solid boxed area in A is enlarged in panels D-F. (C,F) Arrowheads show Caly puncta arrayed along GFP-labeled microtubules in the merged images. Arrows point to concentration of mCh-Caly near the MTOC. Scale bar in A =10 μm. (G) Analyses of overall track or segment displacement, speed, and direction.

Figure 3. Dynein impacts Caly subcellular distribution and movement

A. Distribution of mCh-Caly in Cos-7 cells co-transfected with either tubulin, or the CC1 peptide of the dynactin p150 subunit, both expressed as GFP fusion proteins. B. Microtubule and Caly distribution in GFP-tubulin and mCh-Caly co-transfected cells before application of 10μM nocodazole (Pre-Noco) or after 80 min treatment (Post-Noco). Based on GFP-tubulin distribution, nocodazole disrupted microtubule structure, and also resulted in a cessation of Caly motility (Supplemental video 1,2). C. In fixed cells, Caly colocalized with early endosome (EEA1) and later endosome (Rab7), where α-tubulin stains tubulin polymers. Cells were also co-transfected with mCh-Caly and GFP-Rab5 or GFP-Rab7 to assess Caly motility in EEs and LEs, respectively (Supplemental video 3,4). Scale Bars =10 μm.

Figure 4. Complexes containing Caly and dynein are present in axons

Endogenous DIC (A) and Lis1 (C) co-localized with endogenous Caly puncta in axons extended by adult rat sensory neurons in culture (arrows). GFP-DIC (B) and GFP-Lis1 (D) co-localized with mCh-Caly puncta in DRG axons. Scale Bars=5 μm. (E) Time-lapse data from two-minute recordings of 30 μm axon segments were used to generate the kymographs of particles moving in DRG axons. GFP-DIC and GFP-Lis1 were observed moving with mCh-Caly in the same particle. F. Dynein and Lis1 were pulled down by GST-Caly from brain extracts. Equal amounts of GST and GST-Caly-C were incubated with brain extracts, eluted proteins were probed with anti-DIC, anti-Lis1 and anti-GST antibodies. Only GST-Caly showed the capacity to bind dynein and Lis1.

Figure 5. Dynein motors contribute to the motility of mCh-Caly organelles in DRG axons

(A) Representative kymographs of mCh-Caly organelles moving in axons of neurons expressing scrambled RNA (control), DHC RNAi, or Lis1 RNAi. (B, C) The percent (B) and numbers (C) of 4 classes of organelle movements were determined for 6-12 axons in three different experiments. Retrograde: towards the cell body; Anterograde: away from the cell body; Both: organelles switched directions one or more times; Static: organelles did not move during the recording interval. Total organelles examined: 346 (scr), 237 (DHC RNAi) and 256 (Lis1 RNAi). Significance determined by one-way ANOVA using Tukey’s multiple comparisons post test; (B, C) *** p<0.0001; ** p= 0.0051.

Figure 6. Lis1 and Dynein overexpression increases the motility of mCh-Caly organelles in DRG axons

(A) Kymographs of mCh-Caly organelles in axons overexpressing Lis1 and DIC. (B, C) Overexpression of Lis1 but not DIC increased the percentage of organelles classified as retrograde (numbers above bars is the number of axons analyzed for each condition). (D, E) Overexpression of Lis1 or DIC increased the speeds and run lengths of retrograde motile events. Lis1 increased the speed of anterograde motile events, and both Lis1 and DIC increased run lengths of anterograde motile events. (B) * p<0.017; (D,E) *** p<0.0001; ** p=0.0007.