Subpixel colocalization reveals amyloid precursor protein-dependent kinesin-1 and dynein association with axonal vesicles

Abstract

Intracellular transport of vesicles and organelles along microtubules is powered by kinesin and cytoplasmic dynein molecular motors. Both motors can attach to the same cargo and thus must be coordinated to ensure proper distribution of intracellular materials. Although a number of hypotheses have been proposed to explain how these motors are coordinated, considerable uncertainty remains, in part because of the absence of methods for assessing motor subunit composition on individual vesicular cargos. We developed a robust quantitative immunofluorescence method based on subpixel colocalization to elucidate relative kinesin-1 and cytoplasmic dynein motor subunit composition of individual, endogenous amyloid precursor protein (APP) vesicles in mouse hippocampal cells. The resulting method and data allow us to test a key in vivo prediction of the hypothesis that APP can recruit kinesin-1 to APP vesicles in neuronal axons. We found that APP levels are well-correlated with the amount of the light chain of kinesin-1 (KLC1) and the heavy chain of cytoplasmic dynein (DHC1) on vesicles. In addition, genetic reduction of APP diminishes KLC1 and DHC1 levels on APP cargos. Finally, our data reveal that reduction of KLC1 leads to decreased levels of DHC1 on APP vesicles, suggesting that KLC1 is necessary for the association of DHC1 to these cargos, and help to explain previously reported retrograde transport defects generated when kinesin-1 is reduced.axonal transport | microtubule motor recruitment | Alzheimer's disease M icrotubule-based transport of vesicles and organelles in neurons is coordinated by kinesin (anterograde) and cytoplasmic dynein (retrograde) motor complexes. These motor proteins and their regulators play critical roles in long-distance signaling events in neurons, neuronal regeneration, and in the development of neurodegenerative disease (1-3). Several diseases in humans, such as hereditary spastic paraplegia (4), CharcotMarie-Tooth type 2 (5), and ALS-like motor degeneration (6), have been linked directly to mutations in genes encoding motor proteins. Additionally, disruption of transport is thought to be an early and perhaps causative event in Alzheimer's disease, Parkinson disease, and ALS, where axonal pathologies including abnormal accumulations of proteins and organelles have been routinely observed (1).Kinesin-1 is composed of heavy chain (KHC or KIF5) and light chain (KLC) subunits (7,8). Motor activity is executed by KHC, and cargo binding and regulatory activities are carried out by both KHC and KLC (9-11). Dynein is a large protein complex with six subunits: a large dynein heavy chain (DHC), an intermediate and light-intermediate chain (DIC and DLIC), and three dynein light chains (DLC) (12). Several types of vesicles, including amyloid precursor protein (APP) vesicles, depend upon kinesin-1 (9, 13-17) for their anterograde movement in axons. These studies raised the possibility that APP transport and its influence on APP proteolytic processing play an important role in the development of Alzheim...

Cellular Functions of the Amyloid Precursor Protein from Development to Dementia

“…In addition to adaptor and signaling proteins, APP CTFs are also implicated in interactions with motor proteins such as kinesin, myosin, and dynein (Cottrell et al, 2005;Goldstein, 2012), thereby possibly controlling their own transport (Rodrigues et al, 2012). For example, kinesin-1 localizes to APP-positive axonal vesicles (Szpankowski et al, 2012), and kinesin-1 may interact with APP either directly or indirectly via JIP1 (Chiba et al, 2014;Goldstein, 2012;Kamal et al, 2001;Lazarov et al, 2005). Intriguingly, striking evidence has been reported that JIP1 can directly control anterograde or retrograde directions of APP transport by controlling the activities of kinesin-1 and dynein (Fu and Holzbaur, 2013).…”

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

“…3. For each image in the Z-stack (cargo, motor 1 and motor 2; generated in step 4.2.14) determine the X-Y coordinates and intensity amplitudes for each fluorescent puncta using an established algorithm to fit 2D Gaussians to the point spread function that represents each point source in each of the three channels 16,20,21 ( Figure 2D).…”

“…To obtain the X-Y coordinates, a custom built MATLAB software package called "Motor Colocalization" was developed 16,20 , which incorporates a Gaussian fitting algorithm developed by Jaqaman, Danuser and colleagues [21][22][23] . The software package and detailed instructions for its use can be obtained upon request.…”

“…Fixed cargo and motor images are superimposed onto live movement kymographs to "map" (colocalize) them to the live cargo movement trajectories 16 . To correlate the live movement of cargoes with the association of motor proteins, colocalization is analyzed using a custom made MATLAB software package called "Motor Colocalization" 16,20 . Fluorescently labeled cargoes and motors generate diffraction-limited punctate features that can partially overlap.…”

“…To resolve the position of overlapping puncta, the software first automatically fits Gaussian functions to each point spread function, representing individual fluorescent puncta, to determine their precise X-Y sub-pixel position coordinates and intensity amplitudes [21][22][23] . The positions of motors and cargoes are subsequently compared to each other to determine colocalization 16,20 . Therefore, this method more precisely assigns colocalization between fluorescent puncta as compared to other methods 24 …”