Biochemistry (Moscow) (v.79, #9)

Logistics in the cell: Cargoes and transportation by E. S. Nadezhdina (847-848).
Eukaryotic cells are large and thus require a vesicular transport system. The system involves the formation of membrane transport containers, their short- and long-distance movements, recognition of destination points, and fusion with other membranes. Understanding the molecular mechanisms of these processes is of theoretical and practical significance. This special issue of Biochemistry (Moscow) collects surveys and experimental articles describing various aspects of vesicular transport.
Keywords: vesicular transport; cytoskeleton; cell motility

The signaling and transport systems of eucaryotic cells are tightly interconnected: intracellular transport along microtubules and microfilaments is required to position signaling-pathway components, while signaling molecules control activity of motor proteins and their interaction with tracks and cargoes. Recent data, however, give evidence that active transport is engaged in signaling as a means of signal transduction. This review focuses on this specific aspect of the interaction of two systems.
Keywords: kinesin; dynein; myosin; microtubules; microfilaments; signal transduction

Characterization of Rabaptin-5 γ isoform by E. V. Korobko; S. L. Kiselev; I. V. Korobko (856-864).
Rab GTPases are key regulators of intracellular membrane traffic acting through their effector molecules. Rabaptin-5 is a Rab5 effector in early endosome fusion and connects Rab5- and Rab4-positive membrane compartments owing to its ability to interact with Rab4 GTPase. Recent studies showed that Rabaptin-5 transcript is subjected to extensive alternative splicing, thus resulting in expression of Rabaptin-5 isoforms mostly bearing short deletions in the polypeptide chain. As interactions of a Rab GTPase with different effectors lead to different responses, functional characterization of Rabaptin-5 isoforms becomes an attractive issue. Indeed, it was shown that Rab GTPase effector properties of Rabaptin-5 and its α and δ isoforms are different. This work focused on another Rabaptin-5 isoform, Rabaptin-5γ. Despite its ability to interact with Rab5, endogenously produced Rabaptin-5γ was absent from early endosomes. Rather, it was found to be tightly associated with trans-Golgi network and partially localized to a Rab4-positive membrane compartment. The revealed intracellular localization of Rabaptin-5γ indicates that it is not involved in Rab5-driven events; rather, it functions in other membrane transport steps. Our study signifies the role of alternative splicing in determination of functional activities of Rab effector molecules.
Keywords: intracellular membrane transport; early endosomes; trans-Golgi network; Rab5 small GTPase; Rab4 small GTPase; small GTPase effectors; Rabaptin-5γ

Receptor-mediated endocytosis and cytoskeleton by E. S. Kornilova (865-878).
Receptor-mediated endocytosis is the most specific pathway for macromolecules and macromolecular complexes generally designated as ligands to enter cells. Upon binding to their transmembrane receptors, the ligands enter endocytic vesicles that fuse with each other giving rise to the so-called early endosomes. The sorting of ligand-receptor complexes internalized in these endosomes depends on their nature: metabolic receptors are recycled back to the plasma membrane, while signaling receptors and their ligands (e.g. receptor tyrosine kinases or receptors associated with tyrosine kinase) are delivered to internal vesicles of the multivesicular late endosomes and finally are degraded after interaction with lysosomes. During these processes, endosomes undergo translocation from the cell periphery to the juxtanuclear region, which is accompanied by multiple fusion, invagination, tabulation, and membrane fission events. This review considers modern concepts of the sorting mechanisms of ligand-receptor complexes, the crosstalk between endosomes, microtubules, and actin, and the role of this crosstalk in endosome maturation.
Keywords: endosomes; ligand-receptor complexes; sorting complexes; microtubules; microfilaments; domain organization of endosomes; membrane remodeling

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors by A. I. Fokin; I. B. Brodsky; A. V. Burakov; E. S. Nadezhdina (879-893).
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.
Keywords: dynein; kinesin; endoplasmic reticulum; ERGIC; ERES; fibroblasts; protein kinase

Specific organization of Golgi apparatus in plant cells by M. S. Vildanova; W. Wang; E. A. Smirnova (894-906).
Microtubules, actin filaments, and Golgi apparatus are connected both directly and indirectly, but it is manifested differently depending on the cell organization and specialization, and these connections are considered in many original studies and reviews. In this review we would like to discuss what underlies differences in the structural organization of the Golgi apparatus in animal and plant cells: specific features of the microtubule cytoskeleton organization, the use of different cytoskeleton components for Golgi apparatus movement and maintenance of its integrity, or specific features of synthetic and secretory processes. We suppose that a dispersed state of the Golgi apparatus in higher plant cells cannot be explained only by specific features of the microtubule system organization and by the absence of centrosome as an active center of their organization because the Golgi apparatus is organized similarly in the cells of other organisms that possess the centrosome and centrosomal microtubules. One of the key factors determining the Golgi apparatus state in plant cells is the functional uniformity or functional specialization of stacks. The functional specialization does not suggest the joining of the stacks to form a ribbon; therefore, the disperse state of the Golgi apparatus needs to be supported, but it also can exist “by default”. We believe that the dispersed state of the Golgi apparatus in plants is supported, on one hand, by dynamic connections of the Golgi apparatus stacks with the actin filament system and, on the other hand, with the endoplasmic reticulum exit sites distributed throughout the endoplasmic reticulum.
Keywords: cytoskeleton; microtubules; actin filaments; Golgi apparatus; plants

Dynamic phase microscopy reveals periodic oscillations of endoplasmic reticulum during network formation by T. V. Vyshenskaya; V. P. Tychinsky; D. G. Weiss; S. A. Kuznetsov (907-916).
Dynamic phase microscopy was used to study the dynamic events of formation of the endoplasmic reticulum (ER) in interphase-arrested Xenopus egg extract. We have shown that the ER periodically oscillated in an ATP-dependent manner in the frequency range of 1.6–2.2 Hz, while the tubular membrane network formed in vitro. The spectral density, i.e. the pattern of a given frequency component in the Fourier spectrum, was strongly correlated with the dynamic events during microtubule-dependent and microtubule-independent ER network formation observed by video-enhanced contrast differential interference contrast and fluorescence microscopy. Because the 1.6–2.2 Hz frequency of oscillation during the network formation was detected both in the presence and absence of microtubules, it appears to be an intrinsic ATP-dependent ER membrane property. Several characteristic active and inactive stages of ER network formation were observed both in the presence and absence of microtubules. However, data analysis of these stages indicated that microtubules and dynein motor activity have a strong influence and a cooperative effect on the kinetics of ER formation by controlled fusion reaction.
Keywords: endoplasmic reticulum; membrane fusion; membrane oscillations; microtubules; dynein; Xenopus ; coherent microscopy

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.
Keywords: cytoskeleton; actin; Arp2/3; dynamin; intracellular vesicles; “comet-like tails”

Use of intracellular transport processes for targeted drug delivery into a specified cellular compartment by A. A. Rosenkranz; A. V. Ulasov; T. A. Slastnikova; Y. V. Khramtsov; A. S. Sobolev (928-946).
Targeted drug delivery into the cell compartment that is the most vulnerable to effects of the corresponding drug is a challenging problem, and its successful solution can significantly increase the efficiency and reduce side effects of the delivered therapeutic agents. To accomplish this one can utilize natural mechanisms of cellular specific uptake of macromolecules by receptor-mediated endocytosis and intracellular transport between cellular compartments. A transporting construction combining the components responsible for different steps of intracellular transport is promising for creating multifunctional modular constructions capable of delivering the necessary therapeutic agent into a given compartment of type-specified cells. This review focuses on intracellular transport peculiarities along with approaches for designing such transporting constructions for new, more effective, and safer strategies for treatment of various diseases.
Keywords: targeted drug delivery; intracellular transport; receptor-mediated endocytosis; transport of macromolecules; nuclear import; modular nanotransporters; cancer therapy

During tumor development cancer cells pass through several stages when cell morphology and migration abilities change remarkably. These stages are named epithelial-mesenchymal and mesenchymal-amoeboid transitions. The molecular mechanisms underlying cell motility are changing during these transitions. As result of transitions the cells acquire new characteristics and modes of motility. Cell migration becomes more independent from the environmental conditions, and thus cell dissemination becomes more aggressive, which leads to formation of distant metastases. In this review we discuss the characteristics of each of the transitions, cell morphology, and the specificity of cellular structures responsible for different modes of cell motility as well as molecular mechanisms regulating each transition.
Keywords: epithelial-mesenchymal transition; mesenchymal-amoeboid transition; actin cortex; lamellipodia; filopodia; bleb; small GTPases

Cytoplasmic microtubules are an obligatory component of the cytoskeleton of all types of cells. Microtubules are involved in many cellular processes including directed transport of vesicles and signaling molecules and changes in cell shape during its spreading, polarization, and movement. The intracellular organization of the system of microtubules and their dynamic properties are different in different types of cells because they play a key role in the implementation of a variety of cell and tissue functions, including the regulation of the endothelial barrier function. This review presents an overview of current studies on the properties of endothelial microtubules, their interaction with other components of the cytoskeleton and cell adhesion structures, and the role of microtubules in the regulation of the endothelial barrier function.
Keywords: endothelial barrier function; cytoskeleton; microtubules; dynamics of microtubules