The mechanism responsible for the transport involves a special family of proteins attached to the particles, called motor proteins, which act as small machines that move along the filaments of the cytoskeleton of the cell. The filaments emanate from a structure near the center of the cell called the centrosome and have a common orientation. Some of the motor proteins that attach to the pigmentation particle move in the direction toward the exterior of the cell while others move toward the center of the cell. Which of the proteins are active and which are passive is signaled by the concentration of cAMP. Thus the network of filaments comprising the cytoskeleton not only serve to give a cell its structural support but also form a series of tracks that direct materials transported by motor proteins to specific destinations within the cell. The motor proteins derive their energy from individual molecules of adenosine triphosphate (ATP) by breaking a phosphate bond in a chemical reaction called ATP hydrolysis.
In recent experiments microtubule filaments of the cytoskeleton have been constructed on the surface of a microscope stage and the movements of individual Kinesin motor proteins have been studied in isolation using optical tweezers. Optical tweezers, also referred to as optical traps, use optical forces to manipulate minute specimens. The optical tweezers were used to place individual Kinesin motor proteins on the microtubule track and to apply forces to a particle transported by the motor. The statistical behavior of the motor was then studied for specific load forces applied to the transported particle.
From optical trap experiments information about the elasticity of the tether that connects the motor protein to its cargo, the number of rate limiting steps in the reaction cycle, and other important features about how the motor functions can be obtained. While much is unknown about the detailed mechanisms that allow for the energy of ATP hydrolysis to be converted into directed motion the experimental data places important constraints on theories of how the motor may function.
Aside from Melonosome transport the Kinesin motor protein is involved in many other transport processes. Some examples include the localization of organelles during cell development, the transport of neurotransmitters to the end of axons, and the generation of forces involved in the movement of spindle poles and separation of chromosomes during mitosis.
Kinesin weighs approximately 380 kDa with two heavy (110-130 kDa) chains and two light (60-70 kDa) chains and has about 500 amino acid residues. The protein structure consists of a pair of globular domains that are nearly identical each roughly 10 nanometers in diameter. The secondary structure of the globular domains consists of a layer of beta-sheets sandwiched between a layer of alpha helices. Each globular domain consists of an ATP catalyzing site and a separate microtubule binding site. In the literature the globular domains are commonly referred to as the legs or heads of Kinesin.
The two heads are each connected by a 15 amino acid loop, referred to as the ’’neck linker”, to a long structure that functions as a tether. The tether is comprised of intertwined alpha helices forming a coiled-coil structure roughly 65 nanometers long with an adapter domain at the end. The adapter domain selectively attaches to cargo vesicles equipped with a compatible adapter. The compatibility of the adapters is thought to be used to regulate transport by determining which specific set of motor proteins, destined for different locations within the cell, attach to and transport the materials. An additional structural feature of the stalk is flexible regions that allow for the stalk to double back on itself so that the adapter can bind to the Kinesin motor domain. This is thought to serve as an important regulatory mechanism to inactivate ATP hydrolysis in the motor domain when Kinesin is not bound to a cargo or microtubule.
Cytoskeleton microtubules are the tracks within the cell on which Kinesin moves. Microtubules are long hollow tubes built from dimers of alpha and beta tubulin protein. The dimers when linked end to end form long polymers called protofilaments. Associated with a protofilament is a plus and minus direction. The plus direction by convention corresponds to the direction from an alpha tubulin to beta tubulin subunit. The dimers of the protofilaments have structures that allow for interaction laterally and the formation of protofilament sheets. A typical structure for a microtubule consists of a 13 protofilament sheet rolled into a hollow tube. These microtubules have a diameter of about 24 nanometers and can have a length exceeding 1 micron.
An additional feature of microtubules is the presence of specialized structures that interact with Kinesin. The interaction sites are spaced 8 nanometers apart corresponding to the length of the alpha-beta dimer when measured along the axis of the protofilament. In optical trap and other experiments it has been found that Kinesin moves along only one protofilament of the microtubule in discrete 8 nanometer increments hydrolyzing only 1 ATP molecule per step. It is believed that the walk cycle consists of each head of Kinesin alternately detaching from the microtubule and reattaching further toward the plus end of the microtubule. In experiments however it is observed that occasionally backward steps do occur for the motor.