Quantitative measurements and modeling of cargo–motor interactions during fast transport in the living axon

Abstract

The kinesins have long been known to drive microtubule-based transport of sub-cellular components, yet the mechanisms of their attachment to cargo remain a mystery. Several different cargo-receptors have been proposed based on their in vitro binding affinities to kinesin-1. Only two of these—phosphatidyl inositol, a negatively charged lipid, and the carboxyl terminus of the amyloid precursor protein (APP-C), a trans-membrane protein—have been reported to mediate motility in living systems. A major question is how these many different cargo, receptors and motors interact to produce the complex choreography of vesicular transport within living cells. Here we describe an experimental assay that identifies cargo–motor receptors by their ability to recruit active motors and drive transport of exogenous cargo towards the synapse in living axons. Cargo is engineered by derivatizing the surface of polystyrene fluorescent nanospheres (100 nm diameter) with charged residues or with synthetic peptides derived from candidate motor receptor proteins, all designed to display a terminal COOH group. After injection into the squid giant axon, particle movements are imaged by laser-scanning confocal time-lapse microscopy. In this report we compare the motility of negatively charged beads with APP-C beads in the presence of glycine-conjugated non-motile beads using new strategies to measure bead movements. The ensuing quantitative analysis of time-lapse digital sequences reveals detailed information about bead movements: instantaneous and maximum velocities, run lengths, pause frequencies and pause durations. These measurements provide parameters for a mathematical model that predicts the spatiotemporal evolution of distribution of the two different types of bead cargo in the axon. The results reveal that negatively charged beads differ from APP-C beads in velocity and dispersion, and predict that at long time points APP-C will achieve greater progress towards the presynaptic terminal. The significance of this data and accompanying model pertains to the role transport plays in neuronal function, connectivity, and survival, and has implications in the pathogenesis of neurological disorders, such as Alzheimer’s, Huntington and Parkinson’s diseases.

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development

“…In support of this model, rapid anterograde axonal transport of APP has been clearly demonstrated in cell culture (Koo et al, 1990; Seamster et al, 2012; Szpankowski et al, 2012). In contrast, several recent reports have shown that much of the holoprotein is actually processed into different fragments that are assorted to distinct transport vesicles before they exit the cell body (Muresan et al, 2009; Villegas et al, 2014).…”

Hippocampal to basal forebrain transport of Mn 2+ is impaired by deletion of KLC1, a subunit of the conventional kinesin microtubule-based motor

“…All three KIF5s are expressed in brain, as well as at least two of the light chains, KLC1 and KLC2 (Rahman et al, 1998). Kinesin-1 has been implicated in transport of synaptic vesicles (Sato-Yoshitake et al, 1992), amyloid precursor protein (Kamal et al, 2001; Kamal et al, 2000; Satpute-Krishnan et al, 2006; Seamster et al, 2012), neurofilaments (Li et al, 2012; Xia et al, 2003), post-synaptic neurochemical receptors (Hirokawa et al, 2010), endoplasmic reticulum and Golgi membranes (Lippincott-Schwartz et al, 1995), and mitochondria (Campbell et al, 2014). Defects in kinesin-1 have been implicated in accelerating Alzheimer’s disease (Stokin et al, 2005), activation of axonal stress kinase with enhanced phosphorylation of tau (Falzone et al, 2010) and abnormal proteasome transport and distribution (Otero et al, 2014).…”

Axonal transport and neurodegenerative disease: vesicle-motor complex formation and their regulation

“…121,247 Biophysical evidence indicated that 15aa of the C-terminus of APP was sufficient to promote the transport of APP in squid axons 122 and that APP beads had a high-binding affinity for kinesin-1. 123 In addition, genetic evidence showed that excess human APP or Drosophila APP-like (APPL) and the loss of function of Drosophila APPL resulted in phenotypes similar to motor protein mutants, indicating a function for APP in axonal transport. 124 Interestingly, the C-terminal deletions of human APP or APPL (including the kinesin-binding region) disrupted APP transport and failed to interact functionally with kinesin-1.…”