Recycling of kinesin-1 motors by diffusion after transport

Abstract

Kinesin motors drive the long-distance anterograde transport of cellular components along microtubule tracks. Kinesin-dependent transport plays a critical role in neurogenesis and neuronal function due to the large distance separating the soma and nerve terminal. The fate of kinesin motors after delivery of their cargoes is unknown but has been postulated to involve degradation at the nerve terminal, recycling via retrograde motors, and/or recycling via diffusion. We set out to test these models concerning the fate of kinesin-1 motors after completion of transport in neuronal cells. We find that kinesin-1 motors are neither degraded nor returned by retrograde motors. By combining mathematical modeling and experimental analysis, we propose a model in which the distribution and recycling of kinesin-1 motors fits a "loose bucket brigade" where individual motors alter between periods of active transport and free diffusion within neuronal processes. These results suggest that individual kinesin-1 motors are utilized for multiple rounds of transport.

Figure 1. Kinesin-1 is not rapidly degraded by the proteasome.

(A,B) Differentiated CAD cells were treated with 5 µM lactacystin for the indicated times. (A) Soluble protein lysates were immunoblotted to detect total levels of ubiquitinated proteins (ubiquitin), KHC, JIP1, β-catenin, FEZ1, and tubulin. (B) The average levels of KHC (black bars), ubiquitin (light gray bars) and β-catenin (dark gray bars) were quantified from 6–8 experiments. Error bars indicate SEM. *, p<0.01 as compared to 0 hr. (C,D) Differentiated CAD cells were untreated (top panel) or were treated with 5 µM lactacystin for 6 hrs (bottom panel) then fixed and stained with a monoclonal antibody to endogenous KHC. Scale bar, 15 µm. The average fluorescence intensity of endogenous KHC at neurite tips was quantified (D) in untreated and lactacystin-treated cells. n = 15 cells from two independent experiments for each condition. Error bars indicate SEM. p = 0.055 for treated versus untreated cells.

Figure 2. Kinesin-1 recycling does not depend on cytoplasmic dynein.

(A,B) Differentiated CAD cells were treated with DMSO control for 2 h or with the cytoplasmic dynein inhibitor ciliobrevin A for 0.5 h, 1 h, or 2 h. The cells were then fixed and co-stained with (A) antibodies to the KHC subunit of kinesin-1 and tubulin or (B) antibodies to the kinesin-1 cargo protein JIP1 and tubulin. Yellow arrowheads, KHC or JIP1 at neurite tips. Scale bar, 10 µm. (C,D) Quantification of the average fluorescence intensity of (C) KHC or (D) JIP1 in neurite tips. AU, arbitrary units. For (C), n = 194 cells (DMSO), 186 cells (0.5 h), 158 cells (1 h), or 109 cells (2 h) over two to three independent experiments. For (D), n = 91 cells (DMSO), 84 cells (0.5 h), 95 cells (1 h), or 84 cells (2 h) over three independent experiments. Error bars indicate SEM.

Figure 3. Kinesin-1 returns to the cell body from neurite tips.

Differentiated CAD cells were transfected with a plasmid encoding PAGFP-tagged KHC. 48 hr later, the cells were imaged by confocal microscopy. Representative times series from three different imaging conditions are shown. (A) No photoactivation. GFP images of the entire field of view were collected over time. In the final image, the total level of expressed PAGFP-KHC was determined by photoactivation of the entire field of view (all-activ.). (B) One photoactivation event. GFP images were collected (pre-activ.) and then PAGFP-KHC in one neurite tip (white box) was photoactivated (post-activ.) followed by imaging of GFP fluorescence for the entire field of view over time. In the final image, the total level of expressed PAGFP-KHC was determined by photoactivation of the entire field of view (all-activ.). (C) Multiple photoactivation events. GFP images were collected (pre-activ.) and then PAGFP-KHC in one neurite tip (white box) was photoactivated (post-activ.) followed by imaging of GFP fluorescence for the entire field of view for 4 min. The neurite tip photoactivation and whole field GFP imaging was then repeated 9 times. In the final image, the total level of expressed PAGFP-KHC was determined by photoactivation of the entire field of view (all-activ.). Scale bar, 10 µm. (D) Quantification of the average PAGFP-KHC fluorescence in the cell body over time. At least 7 cells in two to three independent experiments were imaged for each condition. Error bars indicate SEM.

Figure 4. Diffusion of KHC-mCit in neurites.

Differentiated CAD cells expressing (A–C) the control mCit-GST-NUS fusion protein or (D–F) mCit-KHC were imaged by confocal microscopy. Low-expressing cells were chosen for imaging but the brightness of the image was digitally enhanced to aid in visualization. A 30–40 µm stretch in the middle of the neurite (white boxed region) was photobleached and then the fluorescence recovery was monitored over time. (A,D) Representative pre-bleach images. Kymographs of fluorescence recovery in the bleached region (right panels) were generated by drawing a line along the bleached region of the neurite (white box) and plotting the line from each time point down the y-axis. (B,E) Graphs of fluorescence recovery in the bleached regions of the representative cells in (A,D). The fluorescence recovery data (black circles) were fit by an analytical solution (gray lines). (C,F) The average fluorescence recovery for multiple cells expressing (C) mCit-GST-Nus or (F) mCit-KHC. n = 8 cells each.

Figure 5. The distribution of kinesin-1 fits with a Loose Bucket Brigade.

(A–D) Models for kinesin-1 transport and recycling in neuronal processes. In the Diligent Worker model (A), active kinesin-1 motors (green) transport vesicular cargoes to the plus ends of the microtubules (solid green arrow). The motors disengage from the cargo and track (black arrow), assume the inactive autoinhibited conformation (red), and diffuse in the neurite (dotted red arrows). In the Loose Bucket Brigade (B), active kinesin-1 motors (green) transport vesicular cargoes to the plus ends of the microtubules (solid green arrow) but stochastically fall off the cargo (orange arrows). These motors diffuse within the neurite (dotted red arrows) and can reattach to cargoes (green arrows) and again contribute to the transport of vesicles to the plus ends of microtubules. The Hybrid 1 model (C) assumes that kinesin-1 motors can stochastically fall off the cargo (orange arrows) but cannot reattach while diffusing in the neurite (like the Sloppy Bucket Brigade during active transport and the Diligent Worker during recycling). The Hybrid 2 model (D) assumes that kinesin-1 motors do not fall off during transport but can reattach to cargoes while diffusing in the neurite (like the Diligent Worker during active transport and the Sloppy Bucket Brigade during recycling). For each model, computer simulations were used to determine the predicted distribution of kinesin-1 motors in neuronal processes (bottom panels). (E, F) Experimental determination of kinesin-1 distribution in neuronal processes. (E) Differentiated CAD cells or (F) primary hippocampal neurons expressing EGFP (a marker of cell volume) were stained with an antibody to KHC. The fluorescence intensity of KHC (red lines) and EGFP (green lines) was plotted along the length of the neurite or axon (right panels). The fluorescence intensity of KHC represents the spatial distribution of the total kinesin concentration (a sum of concentrations of bound and freely diffusing kinesin-1). (G) The increasing portion of the kinesin-1 profile (red line) in the neuronal process of the CAD cell in (E) was plotted point-by-point as a ratio of the local fluorescence intensities of KHC and EGFP. The distribution fits with an exponential curve (blue line) and is consistent with the Loose Bucket Brigade model.