Plus-End-Tracking Proteins and Their Interactions at Microtubule Ends

Microtubules are cytoskeletal elements that are essential for a large number of intracellular processes, including mitosis, cell differentiation and migration, and vesicle transport. In many cells, the microtubule network is organized in a radial manner, with one end of a microtubule (the minus end) embedded near the nucleus and the other end (the plus end) exploring cytoplasmic space, switching between episodes of growth and shrinkage. Mammalian plus-end-tracking proteins (+TIPs) localize to the ends of growing microtubules and regulate both the dynamic behavior of microtubules as well as the interactions of microtubules with other cellular components. Because of these crucial roles, +TIPs and the mechanisms underlying their association with microtubule ends have been intensively investigated. Results indicate that +TIPs reach microtubule ends by motor-mediated transport or diffusion. Individual +TIP molecules exchange rapidly on microtubule end-binding sites that are formed during microtubule polymerization and that have a slower turnover. Most +TIPs associate with the end-binding (EB) proteins, and appear to require these ‘core’ +TIPs for localization at microtubule ends. Accumulation of +TIPs may also involve structural features of the microtubule end and interactions with other +TIPs. This complexity makes it difficult to assign discrete roles to specific +TIPs. Given that +TIPs concentrate at microtubule ends and that each +TIP binds in a conformationally distinct manner, I propose that the ends of growing microtubules are ‘nano-platforms’ for productive interactions between selected proteins and that these interactions might persist and be functional elsewhere in the cytoplasm than at the microtubule end at which they originated

The protective protein : a multifunctional lysosomal enzyme

This thesis describes the characterization of a lysosomal protein, the 'protective protein', that has at least two functions. On the one hand it protects lysosomal ~galactosidase and neuraminidase from degradation within the lysosome, hence its name. On the other hand it has peptidase and deamidase activities, that could be involved in protein turnover in lysosomes and hormone (in)activation. Degradation is distinguished here from proteolytic processing, although both involve peptide hydrolysis. The first is, however, an aspecific random process, carried out at multiple sites, whereas the second is a highly specific (single) event. Given the putative function of the protective protein it seemed appropriate to start with an overview of intracellular sites of protein degradation, followed by a section on the biogenesis of lysosomes. The introduction ends with a summary on what is known about lysosomal storage disorders, a group of genetic diseases that are due to defects in lysosomal proteins. The protective protein itself is impaired in the rare disorder galactosialidosis and studies on this disease have been the basis for the discovery of the protein and analysis of its functions

The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. [Results]: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. [Conclusions]: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes.This work was supported by the Earth and Life Sciences (ALW) and Medical Sciences (ZonMw) divisions of the Netherlands Organization for Scientific Research (NWO), the Dutch Cancer Society (KWF), the Dutch Cancer Genomics Centre (CGC) and Centre for Biomedical Genetics (CBG), an EC Integrated Project (EuTRACC), and the Spanish Fondo Investigaciones Sanitarias.Peer Reviewe

Microtubule plus-end tracking proteins:novel modulators of cardiac sodium channels and arrhythmogenesis

The cardiac sodium channel NaV1.5 is an essential modulator of cardiac excitability, with decreased NaV1.5 levels at the plasma membrane and consequent reduction in sodium current (INa) leading to potentially lethal cardiac arrhythmias. NaV1.5 is distributed in a specific pattern at the plasma membrane of cardiomyocytes, with localization at the crests, grooves, and T-tubules of the lateral membrane and particularly high levels at the intercalated disc region. NaV1.5 forms a large macromolecular complex with and is regulated by interacting proteins, some of which are specifically localized at either the lateral membrane or intercalated disc. One of the NaV1.5 trafficking routes is via microtubules (MTs), which are regulated by MT plus-end tracking proteins (+TIPs). In our search for mechanisms involved in targeted delivery of NaV1.5, we here provide an overview of previously demonstrated interactions between NaV1.5 interacting proteins and +TIPs, which potentially (in)directly impact on NaV1.5 trafficking. Strikingly, +TIPs interact extensively with several intercalated disc- and lateral membrane-specific NaV1.5 interacting proteins. Recent work indicates that this interplay of +TIPs and NaV1.5 interacting proteins mediates the targeted delivery of NaV1.5 at specific cardiomyocyte subcellular domains, while also being potentially relevant for the trafficking of other ion channels. These observations are especially relevant for diseases associated with loss of NaV1.5 specifically at the lateral membrane (such as Duchenne muscular dystrophy), or at the intercalated disc (for example, arrhythmogenic cardiomyopathy), and open up potential avenues for development of new anti-arrhythmic therapies.</p

CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation

In germinal centres (GC) mature B cells undergo intense proliferation and immunoglobulin gene modification before they differentiate into memory B cells or long-lived plasma cells (PC). GC B-cell-to-PC transition involves a major transcriptional switch that promotes a halt in cell proliferation and the production of secreted immunoglobulins. Here we show that the CCCTC-binding factor (CTCF) is required for the GC reaction in vivo, whereas in vitro the requirement for CTCF is not universal and instead depends on the pathways used for B-cell activation. CTCF maintains the GC transcriptional programme, allows a high proliferation rate, and represses the expression of Blimp-1, the master regulator of PC differentiation. Restoration of Blimp-1 levels partially rescues the proliferation defect of CTCF-deficient B cells. Thus, our data reveal an essential function of CTCF in maintaining the GC transcriptional programme and preventing premature PC differentiation

TAPping into the treasures of tubulin using novel protein production methods

Microtubules are cytoskeletal elements with important cellular functions, whose dynamic behaviour and properties are in part regulated by microtubule-associated proteins (MAPs). The building block of microtubules is tubulin, a heterodimer of α- and β-tubulin subunits. Longitudinal interactions between tubulin dimers facilitate a head-to-tail arrangement of dimers into protofilaments, while lateral interactions allow the formation of a hollow microtubule tube that mostly contains 13 protofilaments. Highly homologous α- and β-tubulin isotypes exist, which are encoded by multi-gene families. In vitro studies on microtubules and MAPs have largely relied on brain-derived tubulin preparations. However, these consist of an unknown mix of tubulin isotypes with undefined post-translational modifications. This has blocked studies on the functions of tubulin isotypes and the effects of tubulin mutations found in human neurological disorders. Fortunately, various methodologies to produce recombinant mammalian tubulins have become available in the last years, allowing researchers to overcome this barrier. In addition, affinity-based purification of tagged tubulins and identification of tubulin-associated proteins (TAPs) by mass spectrometry has revealed the 'tubulome' of mammalian cells. Future experiments with recombinant tubulins should allow a detailed description of how tubulin isotype influences basic microtubule behaviour, and how MAPs and TAPs impinge on tubulin isotypes and microtubule-based processes in different cell types

Cytoplasmic linker proteins promote microtubule rescue in vivo

The role of plus end–tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin

Characterisation of transcriptionally active and inactive chromatin domains in neurons

The tandemly organised ribosomal DNA (rDNA) repeats are transcribed by a dedicated RNA polymerase in a specialised nuclear compartment, the nucleolus. There appears to be an intimate link between the maintenance of nucleolar structure and the presence of heterochromatic chromatin domains. This is particularly evident in many large neurons, where a single nucleolus is present, which is separated from the remainder of the nucleus by a characteristic shell of heterochromatin. Using a combined fluorescence in situ hybridisation and immunocytochemistry approach, we have analysed the molecular composition of this highly organised neuronal chromatin, to investigate its functional significance. We find that clusters of inactive, methylated rDNA repeats are present inside large neuronal nucleoli, which are often attached to the shell of heterochromatic DNA. Surprisingly, the methylated DNA-binding protein MeCP2, which is abundantly present in the centromeric and perinucleolar heterochromatin, does not associate significantly with the methylated rDNA repeats, whereas histone H1 does overlap partially with these clusters. Histone H1 also defines other, centromere-associated chromatin subdomains, together with the mammalian Polycomb group factor Eed. These dat