Microtubule motors in spindle and chromosome motility.

Many of the kinesin microtubule motor proteins discovered during the past 8-9 years have roles in spindle assembly and function or chromosome movement during meiosis or mitosis. The discovery of kinesin motor proteins with a clear involvement in spindle and chromosome motility, together with recent evidence that cytoplasmic dynein plays a role in chromosome distribution, has attracted great interest. The identification of microtubule motors that function in chromosome distribution represents a major advance in understanding the forces that underlie chromosome and spindle movements during cell division.     

Regulation of kinesin-directed movements

Bidirectional organelle transport along microtubules is most likely mediated by the opposing forces generated by two microtubule-based motors: kinesin and cytoplasmic dynein. Because the direction and timing of organelle movements are controlled by the cell, the activity of one or both of these motor molecules must be regulated. Recent studies demonstrate that kinesin, kinesin-like proteins and kinesin-associated proteins can be phosphorylated, and suggest that changes in their phosphorylation state may modulate kinesin's ability to interact with either microtubules or organelles. Thus, it is possible that phosphorylation regulates kinesin-driven movements.     

Motor proteins in cell division

The movements of eukaryotic cell division depend upon the conversion of chemical energy into mechanical work, which in turn involves the actions of motor proteins, molecular transducers that generate force and motion relative cytoskeletal elements. In animal cells, microtubule-based motor proteins of the mitotic apparatus are involved in segregating chromosomes and perhaps in organizing the mitotic apparatus itself, while microfilament-based motors in the contractile ring generate the forces that separate daughter cells during cytokinesis. This review outlines recent advances in our understanding of the roles of molecular motors in mitosis and cytokinesis.     

Cytoplasmic motors and pollen tube growth

The growth of pollen tubes is characterized by an intense cytoplasmic streaming, during which the movements of smaller organelles (like secretory vesicles) and larger ones (including the generative cell and vegetative nucleus) are precisely coordinated. A well-characterized cytoskeletal apparatus is likely responsible for these intracellular movements. In recent years both microfilament and microtubule-based motor proteins have been identified and assumed to be the translocators of the several organelle categories. Their precise function during pollen tube growth is not yet clear, but apparently an actomyosin-based system is mainly responsible for pollen tube elongation. On the other hand, microtubules and microtubule-based motors have been thought to play a role in the maintenance of cell polarity. Both cytoskeletal systems (and their respective motor activities) could cooperate to ensure a precise regulation of pollen tube growth.     

STRUCTURAL AND ULTRASTRUCTURAL FEATURES OF CORTICAL CELLS IN MOTOR ORGANS OF SENSITIVE PLANTS

(1) The movements are only expressed in motor cells, regardless of the nature of the stimulation or its point of application. Therefore, these cells have structures capable of traducing the different stimulation-induced messages which are received in parts incapable of movement. (2) K⁺, Cl^- and Ca²⁺ are the major ions. Their fluxes have been followed during nyctinastic movements as well as during stimuli-induced movements. At the moment, the location and the role of these ions are being studied. (3) The movement results from the integrated activity of all (n) motor cells in the pulvini (i.e. n > 350 × 10³ in primary pulvini 3 mm long and 1·9 mm thick). (4) The motor cell is a full-grown cell whose osmotic activity induces turgor variations allowing foliar movements. (5) The motor cell is a highly differentiated cell, which, up to now, has never been able to dedifferentiate in order to produce callus. (6) The motor cell has original features in its apoplastic compartment (large meatuses, wall foldings, large periplasm with membranes) and in its symplastic compartment (double vacuolar apparatus, morphological polarity given by the tannin vacuole location near the nucleus, abundant mitochondria). (7) Its cytoskeleton includes microtubules, cytoplasmic and vacuolar fibrils (in particular in the tannin vacuole), and a wall with special properties. (8) The motor cell is supposed to contain contractile proteins, whose nature and location are being investigated. (9) The shape change of the motor cell is obvious after pulvinar bending. This change is probably associated with a volume change in several intracellular compartments (vacuole, mitochondria, vesicles). (10) At the cellular and subcellular level the same general features are observed in motor cells of non-seismonastic and of seismonastic species. Probably, functional differences depend upon differences occurring at the molecular level. (11) The motor cell is an interesting model for the study of the osmoregulation mechanism in plant cells, to test the effect of toxic products, in particular to find their optimal efficiency in the circadian cycle.     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization

Background  

Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds.     

Pure thoughts with impure proteins: Permeabilized cell models of organelle motility

Permeabilized cell models provide an experimental middle ground wherein the in vitro properties of mechanochemical proteins can be reconciled with the physical and topological constraints of the intact cell. Several well-studied examples of organelle motility are described here, including the actin-based cytoplasmic streaming of Characean algae, the microtubule-based aggregation and dispersion of pigment granules in chromatophores and the saltatory movements of vesicles along microtubules in fibroblasts and macrophages. The permeabilized models developed for these systems have helped to integrate observations in vivo with in vitro assays of motor proteins.     

Chromobility: the rapid movement of chromosomes in interphase nuclei

There are an increasing number of studies reporting the movement of gene loci and whole chromosomes to new compartments within interphase nuclei. Some of the movements can be rapid, with relocation of parts of the genome within less than 15 min over a number of microns. Some of these studies have also revealed that the activity of motor proteins such as actin and myosin are responsible for these long-range movements of chromatin. Within the nuclear biology field, there remains some controversy over the presence of an active nuclear acto-myosin motor in interphase nuclei. However, both actin and myosin isoforms are localized to the nucleus, and there is a requirement for rapid and directed movements of genes and whole chromosomes and evidence for the involvement of motor proteins in this relocation. The presence of nuclear motors for chromatin movement is thus an important and timely debate to have.     

Putting On The Breaks: Regulating Organelle Movements in Plant Cells†

A striking characteristic of plant cells is that their organelles can move rapidly through the cell. This movement, commonly referred to as cytoplasmic streaming, has been observed for over 200 years, but we are only now beginning to decipher the mechanisms responsible for it. The identification of the myosin motor proteins responsible for these movements allows us to probe the regulatory events that coordinate organelle displacement with normal cell physiology. This review will highlight several recent developments that have provided new insight into the regulation of organelle movement, both at the cellular level and at the molecular level.     

Analysis of microtubule movement on isolated Xenopus egg cortices provides evidence that the cortical rotation involves dynein as well as Kinesin Related Proteins and is regulated by local microtubule polymerisation

In amphibians, the cortical rotation, a translocation of the egg cortex relative to the cytoplasm, specifies the dorsoventral axis. The cortical rotation involves an array of subcortical microtubules whose alignment is mediated by Kinesin-related proteins (KRPs), and stops as M-phase promoting factor (MPF) activation propagates across the egg. To dissect the role of different motor proteins in the cortical rotation and to analyse their regulation, we have developed an open cell assay system involving reactivation of microtubule movement on isolated cortices. Microtubule movements were dependent on ATP and consisted mainly of wriggling and flailing without net displacement, consistent with a tethering of microtubules to the cortex. Reactivated movements were inhibited by anti-KRP and anti-dynein antibodies perfused together but not separately, the KRP antibody alone becoming fixed to the cortex. Neither antibody could inhibit movement in the presence of MPF, indicating that arrest of the cortical rotation is not due to MPF-dependent inhibition of motor molecules. In contrast, D(2)O treatment of live eggs to protect microtubules from progressive depolymerisation prolonged the cortical rotation. We conclude that the cortical rotation probably involves cytoplasmic dynein as well as cortical KRPs and terminates as a result of local MPF-dependent microtubule depolymerisation.     

Neuronal mechanisms of motor signal transmission in the thalamic ventrooral nucleus in spasmodic torticollis patients

A microelectrode technique was used to study the neuronal mechanisms of motor signal transmission in the ventrooral internus nucleus (Voi) of the motor thalamus during voluntary and involuntary pathological (dystonic) movements in patients with spasmodic torticollis. Voi cell elements proved highly reactive to various functional (mostly motor) tests. An activity analysis of 55 Voi neurons detected during nine stereotactic operations revealed, first, a difference in neuronal mechanisms of motor signal transmission for voluntary movements that do or do not involve the affected axial muscles of the neck and for passive and abnormal involuntary dystonic movements. Second, a sensory component was found to play a key role in the mechanisms of sensorimotor interactions during voluntary and involuntary dystonic head and neck movements activating the axial muscles of the neck. Third, rhythmic and synchronized activity of Voi neurons was shown to play an important role in motor signal transmission during voluntary and passive movements. The Voi nucleus was directly implicated in the mechanisms of involuntary head movements and tension of the neck muscles in spasmodic torticollis. The results can be used to identify the Voi nucleus of the thalamus during stereotactic neurosurgery in order to select the optimal destruction or stimulation target and to reduce the postoperative effects in spasmodic torticollis patients.     

Function and regulation of dynein in mitotic chromosome segregation

Cytoplasmic dynein is a large minus-end-directed microtubule motor complex, involved in many different cellular processes including intracellular trafficking, organelle positioning, and microtubule organization. Furthermore, dynein plays essential roles during cell division where it is implicated in multiple processes including centrosome separation, chromosome movements, spindle organization, spindle positioning, and mitotic checkpoint silencing. How is a single motor able to fulfill this large array of functions and how are these activities temporally and spatially regulated? The answer lies in the unique composition of the dynein motor and in the interactions it makes with multiple regulatory proteins that define the time and place where dynein becomes active. Here, we will focus on the different mitotic processes that dynein is involved in, and how its regulatory proteins act to support dynein. Although dynein is highly conserved amongst eukaryotes (with the exception of plants), there is significant variability in the cellular processes that depend on dynein in different species. In this review, we concentrate on the functions of cytoplasmic dynein in mammals but will also refer to data obtained in other model organisms that have contributed to our understanding of dynein function in higher eukaryotes.     

The Zebrafish trilobite gene is essential for tangential migration of branchiomotor neurons

Newborn neurons migrate extensively in the radial and tangential directions to organize the developing vertebrate nervous system. We show here that mutations in zebrafish trilobite (tri) that affect gastrulation-associated cell movements also eliminate tangential migration of motor neurons in the hindbrain. In the wild-type hindbrain, facial (nVII) and glossopharyngeal (nIX) motor neurons are induced in rhombomeres 4 and 6, respectively, and migrate tangentially into r6 and r7 (nVII) and r7 (nIX). In all three tri alleles examined, although normal numbers of motor neurons are induced, nVII motor neurons are found exclusively in r4, and nIX-like motor neurons are found exclusively in r6. The migration of other neuronal and nonneuronal cell types is unaffected in tri mutants. Rhombomere formation and the development of other hindbrain neurons are also unaffected in tri mutants. Furthermore, tangential neuronal migration occurs normally in the gastrulation mutant knypek, indicating that the trilobite neuron phenotype does not arise nonspecifically from aberrant gastrulation-associated movements. We conclude that trilobite function is specifically required for two types of cell migration that occur at different stages of zebrafish development.     

Stepwise movements in vesicle transport of HER2 by motor proteins in living cells

The stepwise movements generated by myosin, dynein, and kinesin were observed in living cells in an attempt to understand the molecular mechanisms of movement within cells. First, the sequential process of the transport of vesicles, including human epidermal factor 2 receptor, after endocytosis was observed for long periods in three dimensions using quantum dots (QDs) and a three-dimensional confocal microscope. QD vesicles, after being endocytosed into the cells, moved along the membrane by transferring actin filaments and were then rapidly transported toward the nucleus along microtubules. Second, the position of vesicles was detected with a precision up to 1.9 nm and 330 micros using a new two-dimensional tracking method. The movement of the QDs transported by myosin VI lying just beneath the cell membrane consisted of 29- and 15-nm steps with a transition phase between these two steps. QD vesicles were then transported toward the nucleus or away from the nucleus toward the cell membrane with successive 8-nm steps. The stepwise movements of these motor proteins in cells were observed using new imaging methods that allowed the molecular mechanisms underlying traffic to and from the membrane to be determined.     

Membrane trafficking, organelle transport, and the cytoskeleton

Cytoskeleton-associated motor proteins typically drive organelle movements in eukaryotic cells in a manner that is tightly regulated, both spatially and temporally. In the past year, a novel organelle transport mechanism utilizing actin polymerization was described. Important advances were also made in the assignment of functions to several new motors and in our understanding of how motor proteins are regulated during organelle transport. In addition, insights were gained into how and why organelles are transported cooperatively along the microtubule and actin cytoskeletons, and into the importance of motor-mediated transport in the organization of the cytoskeleton itself.     

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles — transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles.     

Intracellular transport based on actin polymerization

In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called “comet-like tail”, is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.     

Nucleotide switches in molecular motors: structural analysis of kinesins and myosins

Recent breakthroughs in the structural biology of cytoskeletal motor proteins show that two distinct families of motors--kinesins and myosins - use a similar mechanism of conformational switching for converting small structural changes in their nucleotide-binding sites into larger movements to provide force generation and motion. This mechanism is found to be similar to that employed by G proteins, the well-known molecular switches that regulate protein-protein interactions in many biological systems.     

The kinesin superfamily: tails of functional redundancy

Kinesin is a microtubule-based motility protein that mediates axonal transport and perhaps other intracellular movements in eukaryotic cells. Recent research has indicated that the principal component of kinesin, the kinesin heavy chain, is but one member of an extended superfamily of kinesin-like microtubule motor proteins. These proteins appear to have diverse microtubule-based motility functions--in mitosis, meiosis, vesicle transport and organelle transport. The various kinesin-like molecules may play overlapping or redundant roles in these processes.     

Molecular motors: from one motor many tails to one motor many tales

Kinesin and dynein molecular motor proteins generate the movement of a wide variety of materials in cells. Such movements are crucial for many different cellular and developmental functions, including organelle movement, localization of developmental determinants, mitosis, meiosis and possibly long-range signaling in neurons. Kinesins that control the dynamics of microtubules have also been discovered. Recent work has begun to identify processes in which defective molecular motor function can cause human disease.