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4.15 Intracellular Transport Relating Single-Molecule Properties to in Vivo Function 4.15 Intracellular Transport: Relating Single-Molecule Properties to In Vivo Function GT Shubeita, The University of Texas at Austin, Austin, TX, USA SP Gross, University of California at Irvine, Irvine, CA, USA r 2012 Elsevier B.V. All rights reserved. 4.15.1 Overview 287 4.15.2 An Introduction to the Families of Molecular Motors 287 4.15.3 Single-Molecule Properties of the Molecular Motors 289 4.15.4 From Single Motor Function to In Vivo Transport: The Issues 289 4.15.4.1 How Many Motors Move Cargos In Vivo? 290 4.15.4.2 How Do Multiple Motors Function Together In Vitro? 291 4.15.4.2.1 Motion along a single filament 291 4.15.4.2.2 Motion involving two filaments 293 4.15.4.3 Is Regulation of the Number of Engaged Motors Used to Regulate Transport In Vivo? 294 4.15.4.3.1 Possible regulation via cargo attachment 294 4.15.4.3.2 Possible regulation via filament modification 294 4.15.4.4 Do Motors In Vivo Function the Same Way as They Do In Vitro? 295 4.15.5 Summary 296 References 296 Abbreviations LD lipid droplet ATP adenosine triphosphate MAP microtubule-associated protein EM electron microscopy Glossary Microtubule A stiff polymeric filament in cells used as a Dynein A motor protein that moves in the opposite highway for long-distance transport; the motors kinesin and direction from kinesin along microtubules. dynein ‘walk’ along it, with kinesin moving toward the Kinesin An enzymatic protein known as a motor protein microtubule plus end and dynein moving toward the minus because it hydrolyzes ATP and converts the energy stored in end. the phosphate bond to motion. 4.15.1 Overview single-molecule level have been developed motivated by the desire to understand the proteins mechanistically, at a fun- In older textbooks, there is little mention of how cells damental level. There are three such classes of motors – the are organized and the importance of that organization. The myosin motors that move along actin filaments, the kinesin modern view is quite different. We now know that many motors that move along microtubules, and the dynein motors cellular processes depend on the existence of distinct sub- that also move along microtubules – and reviews describe cellular compartments with unique properties and on the what we know about the motors’ single-molecule functions.1,2 spatial organization of these compartments relative to each Thus, we know much about the way these proteins function, other, allowing controlled transport of components between although there are still interesting open questions related to them. Both the positioning of these compartments and the the mechanism. transport between them rely on enzymes called molecular However, single-molecule knowledge is only the start of motors that walk along cytoskeletal tracks. These motors are this journey to understand cellular function and organ- true nanomachines: They burn fuel (ATP) and use this ization. We cannot easily explain how transport and or- chemical energy to do mechanical work, taking 8- to 36-nm ganization processes in cells work using only the steps (depending on the motor) and exerting forces up to information gained from single-molecule in vitro studies approximately 7 pN. Typically, they are approximately 50% on purified proteins. The challenge before us, then, is to efficient – that is, approximately 50% of the energy released understand how to make this transition. This chapter dis- from ATP hydrolysis can be converted into useful mechanical cusses our current understanding of this process, high- work. lighting progress to date, and the challenges involved in The function of these motors is fascinating, and in no using single-molecule function to understand in vivo small measure the tools to study these proteins at the transport. Comprehensive Biophysics, Volume 4 doi:10.1016/B978-0-12-374920-8.00418-5 287 288 Intracellular Transport: Relating Single-Molecule Properties to In Vivo Function 4.15.2 An Introduction to the Families of Molecular microtubules’ plus ends and the dynein family of motors that Motors moves toward the microtubules’ minus ends. Given typical microtubule organization, one might imagine that transport Many families of molecular motors accomplish various roles of components away from the nucleus would be mediated by in cells. This chapter focuses on cargo transport motors – that kinesin-powered transport, and transport toward the nucleus is, on motors used to move cargos from one subcellular lo- would be mediated by dynein. This is predominantly correct, cation to another. Such cargos can include ‘traditional’ although it is not nearly as straightforward as would initially membrane-bound organelles, such as axonal vesicles or mi- be expected. tochondria, but can also include LDs or protein particles such One interesting aspect of the kinesin and dynein motors is as signaling complexes or viruses, mRNA particles, and ‘large’ that they appear to have taken a slightly different evolutionary cargos such as chromosomes or entire nuclei. Long-distance strategy. There are many kinesin family member proteins, each transport occurs along microtubules, which are polar fila- likely optimized for moving a subset of intracellular cargos.3 ments with a ‘plus’ end and a ‘minus’ end. The microtubules Almost all kinesin motors move toward the microtubule plus are usually arranged with their minus end close to the nucleus end, although there are a few slow mitotic kinesin motors that and their plus end away from the nucleus. For many cells move toward the minus end. In contrast, there are only a few with the nucleus in the cell center, this corresponds to minus cytoplasmic dynein motors (all moving toward the micro- end at the cell center and plus end at the periphery. However, tubule minus end), and these few motors each move many other organizations of microtubules are frequently found different kinds of cargos. Accumulating evidence suggests that in polarized cells (Figure 1). There are two classes of for dynein, much of the ‘tailoring’ of the motor for specific motors (Figure 2) that move along microtubules: the kinesin tasks is accomplished by mixing and matching different family of motors that predominantly moves toward the dynein complex subunits and accessory proteins.4 Although such a hypothesis is appealing, it is still far from proven, and certainly more experimental data on the effects of protein cofactors will be required to evaluate the extent of difference in ‘tunability’ between dynein and kinesin. Microtubules are long, typically with a rather well-defined organization. In contrast, actin filaments – used by the myosin family of molecular motors – are shorter and have much more variation in their organization. As such, it is clear that actin/ myosin-based transport in typical mammalian cells cannot be responsible for the majority of long-distance transport, and (a) Actin filament Cargo (b) (c) Myosin Figure 1 Different cell types have different microtubule architectures. Long-distance transport of organelles and other cargos Dynein occurs along microtubules (shown in red), which are filaments with directionality, typically indicated by referring to the ‘plus’ end ( þ ) Kinesin and the ‘minus’ end ( À ). The microtubules are usually arranged with their minus ends close to the nucleus and their plus ends away from − + the nucleus. In many cell types, the nucleus is in the cell center Fig. Minus Plus 2 in the International Journal of Biochemistry & Cell biology 40(4), Microtubule end end 2008, pages 619–637 (b), but other organizations are possible, as in the case of the Drosophila embryo schematized in panel c. Given the Figure 2 Molecular motors attach to both cargos and filaments. high aspect ratio of neuronal cell processes (a), motor-based Cargos are hauled inside cells by molecular motors along cytosolic transport of cargos is especially vital. Microtubules in the axons are filaments. Two families of microtubule motors, kinesins and typically arranged with their minus ends all pointing toward the cell cytoplasmic dynein, are responsible for most long-distance transport. body and their plus ends away from it (right insert in panel a). Kinesins and dyneins predominantly move toward the plus ends and Dendrites, on the other hand, have shorter microtubules that have a minus ends of microtubules, respectively. Members of the myosin mixed organization Fig. 1 from phys. Biol 1 (2004) RI-RII, Hithes and families of motors move cargos along actin filaments and translocate yon: a review of bi-directional microtubule-based transport, steven P. toward either the barbed ends or the pointed ends of the filaments, Gross (left insert in panel a). depending on the myosin isoform. Intracellular Transport: Relating Single-Molecule Properties to In Vivo Function 289 any description of transport involving them must carefully Building complexity consider the actin filament organization as well as properties of the motors. However, there is significant evidence that in some cases actin-based transport is used as a local road system for transport to places that microtubules do not reach.5–7 Also, there is surprising work demonstrating long-distance actin/ myosin-based transport in special cellular projections where actin is more organized.8 Single motor Because actin filaments are also polar/directed, one might expect there to be myosin family motors that move in each direction along them. This is indeed correct, with almost all myosins, including the myosin V and myosin X family motors, moving toward the barbed ( þ ) end, and the myosin VI families moving toward the pointed ( À ) end of the filaments. 4.15.3 Single-Molecule Properties of the Molecular Motors Biophysical approaches have been successful at determining 1,2 how single molecular motors function. Motors are charac- Multiple motors terized by their processivity (how far a motor typically walks along its filament before detaching), their stalling force, and their velocity.
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