Bruce J. Schnapp

A Change in the Selective Translocation of the Kinesin-1 Motor Domain Marks the Initial Specification of the Axon

We used the accumulation of constitutively active kinesin motor domains as a measure of where kinesins translocate in developing neurons. Throughout development, truncated Kinesin-3 accumulates at the tips of all neurites. In contrast, Kinesin-1 selectively accumulates in only a subset of neurites. Before neurons become polarized, truncated Kinesin-1 accumulates transiently in a single neurite. Coincident with axon specification, truncated Kinesin-1 accumulates only in the emerging axon and no longer appears in any other neurite. The translocation of Kinesin-1 along a biochemically distinct track leading to the nascent axon could ensure the selective delivery of Kinesin-1 cargoes to the axon and hence contribute to its molecular specification. Imaging YFP-tagged truncated Kinesin-1 provides the most precise definition to date of when neuronal polarity first emerges and allows visualization of the molecular differentiation of the axon in real time.

The Kinesin Motor KIF3A Is a Component of the Presynaptic Ribbon in Vertebrate Photoreceptors

Kinesin motors are presumed to transport various membrane compartments within neurons, but their specific in vivofunctions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.

Dynactin-Dependent, Dynein-Driven Vesicle Transport in the Absence of Membrane Proteins: A Role for Spectrin and Acidic Phospholipids

We reconstituted dynein-driven, dynactin-dependent vesicle transport using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, we show that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. We verify this model in the context of liposomes, isolated axonal vesicles, and whole axoplasm. We conclude that spectrin has an essential role in retrograde axonal transport.

Trafficking of signaling modules by kinesin motors

The human genome has more than 40 kinesin genes whose protein products organize intracellular traffic along microtubules. Research during the past two years has begun to elucidate the cargoes carried by kinesins and the nature of the kinesin-cargo linkage. Modular protein-protein interactions connect kinesins to diverse cellular molecules, which, apart from their other functions, serve as kinesin-cargo linkers. Many of these newly identified linkers are scaffolds for signaling pathways, and mounting evidence now indicates that kinesins transport pre-assembled signaling modules as vesicular cargo. These findings bring together two fields, signal transduction and molecular motors, and lead to a deeper understanding of the interplay between trafficking, localization and intercellular communication.

Zebrafish melanophilin facilitates melanosome dispersion by regulating dynein.

BACKGROUND Fish melanocytes aggregate or disperse their melanosomes in response to the level of intracellular cAMP. The role of cAMP is to regulate both melanosome travel along microtubules and their transfer between microtubules and actin. The factors that are downstream of cAMP and that directly modulate the motors responsible for melanosome transport are not known. To identify these factors, we are characterizing melanosome transport mutants in zebrafish. RESULTS We report that a mutation (allele j120) in the gene encoding zebrafish melanophilin (Mlpha) interferes with melanosome dispersion downstream of cAMP. Based on mouse genetics, the current model of melanophilin function is that melanophilin links myosin V to melanosomes. The residues responsible for this function are conserved in the zebrafish ortholog. However, if linking myosin V to melanosomes was Mlphas sole function, elevated cAMP would cause mlpha(j120) mutant melanocytes to hyperdisperse their melanosomes. Yet this is not what we observe. Instead, mutant melanocytes disperse their melanosomes much more slowly than normal and less than halfway to the cell margin. This defect is caused by a failure to suppress minus-end (dynein) motility along microtubules, as shown by tracking individual melanosomes. Disrupting the actin cytoskeleton, which causes wild-type melanocytes to hyperdisperse their melanosomes, does not affect dispersion in mutant melanocytes. Therefore, Mlpha regulates dynein independently of its putative linkage to myosin V. CONCLUSIONS We propose that cAMP-induced melanosome dispersion depends on the actin-independent suppression of dynein by Mlpha and that Mlpha coordinates the early outward movement of melanosomes along microtubules and their later transfer to actin filaments.

RNA localization: A glimpse of the machinery

Asymmetric mRNA localization within cells plays an important part in both development and physiology. Recent studies have provided a glimpse of the conserved molecular machinery that directs the localization of specific mRNAs.

RNA localization: SHEdding light on the RNA–motor linkage

Specific mRNAs achieve an asymmetric distribution in the cell by linking to molecular motors that walk along the cytoskeleton. Studies in S. cerevisiae have begun to define the nature of the RNA-motor linkage and identify She3p as an adaptor protein that links a type V myosin motor to specific ribonucleoproteins.

Improved nm displacement detector for microscopic beads at frequencies below 10 Hz

A modular differential interference contrast microscope combined with laser interferometry has been designed and constructed to detect nm-scale displacements of microscopic beads with a root mean square noise less than 0.4 nm. The unique feature of our system lies in its capability of preserving the nm-scale detection sensitivity down to a low-frequency region of a few Hz. The modularized design ensures the system is accessible to further modifications as frequently required by various biological experiments such as employing multiple laser traps to manipulate rod-shaped samples.

Projecting two-axis nanometer scale displacement of microscopic beads onto a quadrant photodetector with a laser beam

A quadrant photodetector has been added onto a custom differential interference contrast microscope to measure two-axis displacement of silica beads with a noise ⩽0.1 nm/√Hz below 150 Hz. A diode-pumped solid-state laser is incorporated to function not only as a light source for displacement detection but also as an optical trap for motility studies of protein motors. This system is designed for studies in which the motility needs to be simultaneously monitored along both axes at nm accuracy.

Nanometer measurements of motor protein and membrane glycoprotein movements

The recent application of digital imaging technologies to the analysis of video images from a DIC (differential interference contrast) light microscope has given 1-2 nm precision for the measurement of movements of submicron particles at 30 Hz. [Gelles et al., Nature 331(1988)450]. This technique has been applied to the study of the movements of 1) motor proteins (kinesin and cytoplasmic dynein) on microtubules (MTs) and 2) membrane glycoproteins in cellular plasma membranes. In both cases the nanometer measurements have provided important new insights into the molecular mechanisms which govern those biological movements.