The central tubuli in distal segments of olfactory cilia lack dynein arms

Summary By means of the tannic acid-glutaraldehyde fixation method, the lack of dynein bridges between the central two tubuli in distal segments of mouse olfactory cilia is demonstrated. Consequently, these organelles are supposed to be unable to beat actively, in contrast to the proximal ciliary shafts.Supported by grant Nr. 2099 from Fonds zur Förderung der wissenschaftlichen Forschung.     

The central tubuli in distal segments of olfactory cilia lack dynein arms.

By means of the tannic acid-glutaraldehyde fixation method, the lack of dynein bridges between the central two tubuli in distal segments of mouse olfactory cilia is demonstrated. Consequently, these organelles are supposed to be unable to beat actively, in contrast to the proximal ciliary shafts.     

Light-dependent phosphorylation of Bardet–Biedl syndrome 5 in photoreceptor cells modulates its interaction with arrestin1

Arrestins are dynamic proteins that move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the light conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1. Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that Bardet–Biedl syndrome 5 (BBS5) is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions. Our principal findings in this study are threefold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed.     

Microtubule transport defects in neurological and ciliary disease

Microtubules are primarily responsible for facilitating long-distance transport of both proteins and organelles. Given the critical role of this process in cellular function, it is not surprising that perturbation of microtubule-based transport can lead to diverse phenotypes in humans, including cancer and neurodegenerative disorders such as Alzheimer or Huntington disease. Recent investigations have also indicated that defects in specialized microtubule-based transport systems, such as mutations affecting the transport of protein particles along the length of cilia (intraflagellar transport) can cause retinal dystrophy, polycystic kidney disease or more complex syndromic phenotypes, such as Bardet-Biedl syndrome. In this review, we discuss recent findings implicating defects in microtubule-associated transport and motor proteins in a variety of diseases, particularly the role of defective microtubular transport in neurological and ciliary disease. These defects frequently display phenotypic consequences that manifest as human disease yet do not cause organismal lethality.Received 7 Janury 2005; received after revision 23 February 2005; accepted 21 March 2005     

Structure and assembly of the 20S proteasome

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain α-type subunits, the inner ones comprise β-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the α-type and β-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the β-type subunits are processed. The prosequence of the β-type subunits is essential for the assembly process and prevents protease activity of immature proteasomes.     

Transport and diffusion of Tau protein in neurons

In highly polarized and elongated cells such as neurons, Tau protein must enter and move down the axon to fulfill its biological task of stabilizing axonal microtubules. Therefore, cellular systems for distributing Tau molecules are needed. This review discusses different mechanisms that have been proposed to contribute to the dispersion of Tau molecules in neurons. They include (1) directed transport along microtubules as cargo of tubulin complexes and/or motor proteins, (2) diffusion, either through the cytosolic space or along microtubules, and (3) mRNA-based mechanisms such as transport of Tau mRNA into axons and local translation. Diffusion along the microtubule lattice or through the cytosol appear to be the major mechanisms for axonal distribution of Tau protein in the short-to-intermediate range over distances of up to a millimetre. The high diffusion coefficients ensure that Tau can distribute evenly throughout the axonal volume as well as along microtubules. Motor protein-dependent transport of Tau dominates over longer distances and time scales. At low near-physiological levels, Tau is co-transported along with short microtubules from cell bodies into axons by cytoplasmic dynein and kinesin family members at rates of slow axonal transport.     

Integrase, LEDGF/p75 and HIV replication

HIV integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The essential DNA cleaving and joining chemistry of integration is known, but there is less understanding of the process as it occurs in a cell, where two complex and dynamic macromolecular entities are joined: the viral pre-integration complex and chromatin. Among implicated cellular factors, much recent attention has coalesced around LEDGF/p75, a nuclear protein that may act as a chromatin docking factor or receptor for lentiviral pre-integration complexes. LEDGF/p75 tethers HIV integrase to chromatin, protects it from degradation, and strongly influences the genome-wide pattern of HIV integration. Depleting the protein from cells and/or over-expressing its integrase-binding domain blocks viral replication. Current goals are to establish the underlying mechanisms and to determine whether this knowledge can be exploited for antiviral therapy or for targeting lentiviral vector integration in human gene therapy. Received 25 November 2007; received after revision 7 January 2008; accepted 10 January 2008     

Failure of cilia of reprogram following segmental ampullary reversal of the rabbit oviduct

Summary Cilia exhibited unidirectional and coordinated movement within microsurgically reversed segments of rabbit ampulla when examined up to 13 months after surgery. The direction of ciliary beating was opposite that of the remainder of the oviduct.This research was supported in part by a grant (HD 09339-06) from the National Institutes of Health and the Bioassay and Smooth Muscle Core Laboratories (NIH grant P30 HD10202).     

Etude ultrastructurale de la transformation du corps élémentaire en corps initial chez lesRickettsiella

Summary An intermediate stage between the elementary and the initial bodies of theRickettsiella genus is defined as the beginning of an intracellular cycle. It is characterized by several structural changes in the dense elementary body: the cytoplasm becomes less electron-dense; thus, the nucleoid and the ribosomes are visible. The inner layer of the cell-wall becomes progressively clearer and the trilamellar structure of the inner and outer membranes appears distinctly. Preinitial body is proposed as name of this stage of development.     

Spiralig angeordnete Untereinheiten in den A-Filamenten der Kamptozoenmuskulatur

Summary Thick A-Filaments (myosin filaments) of entoproctan muscle cells each consist of 9–11 fibrillar subunits, ca. 30 å in diameter, embedded in a protein matrix of lower electron density (tropomyosin ?). Unlike hitherto described paramyosin filaments, these subunits are regularly arranged in a single circle near the outer edge of each filament. They seem to run in spiral windings around the filaments axis. The protein matrix shows a faint banding along the filament, resembling to the tropomyosin-A pattern but with a much shorter periodicity (ca. 60 å).

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Mit UnterstÜtzung der Deutschen Forschungsgemeinschaft.     

The biogenesis and function of eukaryotic porins

Summary Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial, protein import with a few aberrations: a) porin contains an,uncleaved NH₂-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential , although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involved at least one receptor protein.Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

NDE1 and NDEL1 from genes to (mal)functions: parallel but distinct roles impacting on neurodevelopmental disorders and psychiatric illness

NDE1 (Nuclear Distribution Element 1, also known as NudE) and NDEL1 (NDE-Like 1, also known as NudEL) are the mammalian homologues of the fungus nudE gene, with important and at least partially overlapping roles for brain development. While a large number of studies describe the various properties and functions of these proteins, many do not directly compare the similarities and differences between NDE1 and NDEL1. Although sharing a high degree structural similarity and multiple common cellular roles, each protein presents several distinct features that justify their parallel but also unique functions. Notably both proteins have key binding partners in dynein, LIS1 and DISC1, which impact on neurodevelopmental and psychiatric illnesses. Both are implicated in schizophrenia through genetic and functional evidence, with NDE1 also strongly implicated in microcephaly, as well as other neurodevelopmental and psychiatric conditions through copy number variation, while NDEL1 possesses an oligopeptidase activity with a unique potential as a biomarker in schizophrenia. In this review, we aim to give a comprehensive overview of the various cellular roles of these proteins in a “bottom-up” manner, from their biochemistry and protein–protein interactions on the molecular level, up to the consequences for neuronal differentiation, and ultimately to their importance for correct cortical development, with direct consequences for the pathophysiology of neurodevelopmental and mental illness.     

The biogenesis and function of eukaryotic porins.

Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial protein import with a few aberrations: a) porin contains an uncleaved NH2-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential delta psi, although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involves at least one receptor protein. Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

Microtubule dynamics alter the interphase nucleus

Microtubules are known to drive chromosome movements and to induce nuclear envelope breakdown during mitosis and meiosis. Here we show that microtubules can enforce nuclear envelope folding and alter the levels of nuclear envelope-associated heterochromatin during interphase, when the nuclear envelope is intact. Microtubule reassembly, after chemically induced depolymerization led to folding of the nuclear envelope and to a transient accumulation of condensed chromatin at the site nearest the microtubule organizing center (MTOC). This microtubule-dependent chromatin accumulation next to the MTOC is dependent on the composition of the nuclear lamina and the activity of the dynein motor protein. We suggest that forces originating from simultaneous polymerization of microtubule fibers deform the nuclear membrane and the underlying lamina. Whereas dynein motor complexes localized to the nuclear envelope that slide along the microtubules transfer forces and/or signals into the nucleus to induce chromatin reorganization and accumulation at the nuclear membrane folds. Thus, our study identified a molecular mechanism by which mechanical forces generated in the cytoplasm reshape the nuclear envelope, alter the intranuclear organization of chromatin, and affect the architecture of the interphase nucleus.     

The role of kinesin, dynein and microtubules in pancreatic secretion

The regulated secretion of pancreatic zymogens depends on a functional cytoskeleton and intracellular vesicle transport. To study the dynamics of tubulin and its motor proteins dynein and kinesin during secretion in pancreatic acinar cells, we infused rats with 0.1 μg/kg/h caerulein. Electron and fluorescence microscopy detected neither dynein nor kinesin at the apical secretory pole, nor on the surface of mature zymogen granules. After 30 min of secretagogue stimulation, kinesin and the Golgi marker protein 58 K were reallocated towards the apical plasma membrane and association of kinesin with tubulin was enhanced. Disruption of acinar cell microtubules had no effect on initial caerulein-induced amylase release but completely blocked secretion during a second stimulus. Our results suggest that mature zymogen granule exocytosis is independent of intact microtubules, kinesin and dynein. However, microtubule-dependent mechanisms seem to be important for the replenishment of secretory vesicles by redistribution of Golgi elements towards the apical cell pole. J. Schnekenburger and I.-A. Weber have contributed equally to this work.     

Regulation of HERG (KCNH2) potassium channel surface expression by diacylglycerol

The HERG (KCNH2) channel is a voltage-sensitive potassium channel mainly expressed in cardiac tissue, but has also been identified in other tissues like neuronal and smooth muscle tissue, and in various tumours and tumour cell lines. The function of HERG has been extensively studied, but it is still not clear what mechanisms regulate the surface expression of the channel. In the present report, using human embryonic kidney cells stably expressing HERG, we show that diacylglycerol potently inhibits the HERG current. This is mediated by a protein kinase C-evoked endocytosis of the channel protein, and is dependent on the dynein–dynamin complex. The HERG protein was found to be located only in early endosomes and not lysosomes. Thus, diacylglycerol is an important lipid participating in the regulation of HERG surface expression and function.     

Structural and functional relationships between photoreceptor tetraspanins and other superfamily members

The two primary photoreceptor-specific tetraspanins are retinal degeneration slow (RDS) and rod outer segment membrane protein-1 (ROM-1). These proteins associate together to form different complexes necessary for the proper structure of the photoreceptor outer segment rim region. Mutations in RDS cause blinding retinal degenerative disease in both rods and cones by mechanisms that remain unknown. Tetraspanins are implicated in a variety of cellular processes and exert their function via the formation of tetraspanin-enriched microdomains. This review focuses on correlations between RDS and other members of the tetraspanin superfamily, particularly emphasizing protein structure, complex assembly, and post-translational modifications, with the goal of furthering our understanding of the structural and functional role of RDS and ROM-1 in outer segment morphogenesis and maintenance, and our understanding of the pathogenesis associated with RDS and ROM-1 mutations.     

Receptor binding, internalization, and retrograde transport of neurotrophic factors

This review deals with the receptor interactions of neurotrophic factors, focusing on the neurotrophins of the nerve growth factor (NGF) family, the glial cell derived neurotrophic factor (GDNF) family, and the ciliary neurotrophic factor (CNTF) family. The finding that two proteins, p75^NTR and Trk, act as receptors for NGF in neurons generated the discovery of other neurotrophic factors/receptor families and has enhanced our understanding of the development, survival, regeneration, and degeneration of the nervous system. The kinetics of binding, the structure of the ligand-receptor complex, and the mechanism of retrograde transport of the neurotrophins are discussed in detail and compared to information available on the GDNF and CNTF families. Each neurotrophic factor family, i.e., NGF, GDNF, and CNTF, has a set of receptors with specificity for individual members of the family and a common receptor without member specificity that, in some families, generates the cellular signal and retrograde transport.