19 research outputs found
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
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
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
Dual effect of CTCF loss on neuroprogenitor differentiation and survival
An increasing number of proteins involved in genome organization have been implicated in neurodevelopmental disorders, highlighting the importance of chromatin architecture in the developing CNS. The CCCTC-binding factor (CTCF) is a zinc finger DNA binding protein involved in higher-order chromatin organization, and mutations in the human CTCF gene cause an intellectual disability syndrome associated with microcephaly. However, information on CTCF function in vivo in the developing brain is lacking. To address this gap, we conditionally inactivated the Ctcf gene at early stages of mouse brain development. Cre-mediated Ctcf deletion in the telencephalon and anterior retina at embryonic day 8.5 triggered upregulation of the p53 effector PUMA (p53 upregulated modulator of apoptosis), resulting in massive apoptosis and profound ablation of telencephalic structures. Inactivation of Ctcf several days later at E11 also resulted in PUMA upregulation and increased apoptotic cell death, and the Ctcf-null forebrain was hypocellular and disorganized at birth. Although deletion of both Ctcf and Puma in the embryonic brain efficiently rescued Ctcf-null progenitor cell apoptosis, it failed to improve neonatal hypocellularity due to decreased proliferative capacity of rescued apical and outer radial glia progenitor cells. This was exacerbated by an independent effect of CTCF loss that resulted in depletion of the progenitor pool due to premature neurogenesis earlier in development. Our findings demonstrate that CTCF activities are required for two distinct events in early cortex formation: first, to correctly regulate the balance between neuroprogenitor cell proliferation and differentiation, and second, for the survival of neuroprogenitor cells, providing new clues regarding the contributions of CTCF in microcephaly/intellectual disability syndrome pathologies
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
The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains
Murine ZFP-37 is a member of the large family of C2H2 type zinc finger
proteins. It is characterized by a truncated NH2-terminal
Kruppel-associated box and is thought to play a role in transcriptional
regulation. During development Zfp-37 mRNA is most abundant in the
developing central nervous system, and in the adult mouse expression is
restricted largely to testis and brain. Here we show that at the protein
level ZFP-37 is detected readily in neurons of the adult central nervous
system but hardly in testis. In brain ZFP-37 is associated with nucleoli
and appears to contact heterochromatin. Mouse and human ZFP-37 have a
basic histone H1-like linker domain, located between KRAB and zinc finger
regions, which binds double-stranded DNA. Thus we suggest that ZFP-37 is a
structural protein of the neuronal nucleus which plays a role in the
maintenance of specialized chromatin domains
Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein)
Several microtubule binding proteins, including CLIP-170 (cytoplasmic
linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been
shown to associate specifically with the ends of growing microtubules in
non-neuronal cells, thereby regulating microtubule dynamics and the
binding of microtubules to protein complexes, organelles, and membranes.
When fused to GFP (green fluorescent protein), these proteins, which
collectively are called +TIPs (plus end tracking proteins), also serve as
powerful markers for visualizing microtubule growth events. Here we
demonstrate that e
Choice of binding sites for CTCFL compared to CTCF is driven by chromatin and by sequence preference
The two paralogous zinc finger factors CTCF and CTCFL differ in expression such that CTCF is ubiquitously expressed, whereas CTCFL is found during spermatogenesis and in some cancer types in addition to other cell types. Both factors share the highly conserved DNA binding domain and are bound to DNA sequences with an identical consensus. In contrast, both factors differ substantially in the number of bound sites in the genome. Here, we addressed the molecular features for this binding specificity. In contrast to CTCF we found CTCFL highly enriched at 'open' chromatin marked by H3K27 acetylation, H3K4 di- and trimethylation, H3K79 dimethylation and H3K9 acetylation plus the histone variant H2A.Z. CTCFL is enriched at transcriptional start sites and regions bound by transcription factors. Consequently, genes deregulated by CTCFL are highly cell specific. In addition to a chromatin-driven choice of binding sites, we determined nucleotide positions critical for DNA binding by CTCFL, but not by CTCF
CTCF induces histone variant incorporation, erases the H3K27me3 histone mark and opens chromatin
Insulators functionally separate active chromatin domains frominactive ones. The insulator factor, CTCF, has been found to bind to boundaries and to mediate insulator function. CTCF binding sites are depleted for the histone modification H3K27me3 and are enriched for the histone variant H3.3. In order to determine whether demethylation of H3K27me3 and H3.3 incorporation are a requirement for CTCF binding at domain boundaries or whether CTCF causes these changes, we made use of the LacI DNA binding domain to control CTCF binding by the Lac inducer IPTG. Here we show that, in contrast to the related factor CTCFL, the N-terminus plus zinc finger domain of CTCF is sufficient to open compact chromatin rapidly. This is preceded by incorporation of the histone variant H3.3, which thereby removes the H3K27me3 mark. This demonstrates the causal role for CTCF in generating the chromatin features found at insulators. Thereby, spreading of a histone modification from one domain through the insulator into the neighbouring domain is inhibited
Distinct Functions for Mammalian CLASP1 and-2 During Neurite and Axon Elongation
Mammalian cytoplasmic linker associated protein 1 and -2 (CLASP1 and -2) are
microtubule (MT) plus-end tracking proteins that selectively stabilize MTs at the
edge of cells and that promote MT nucleation and growth at the Golgi, thereby
sustaining cell polarity. In vitro analysis has shown that CLASPs are MT growth
promoting factors. To date, a single CLASP1 isoform (called CLASP1α) has been
described, whereas three CLASP2 isoforms are known (CLASP2α, -β, and -γ).
Although CLASP2β/γ are enriched in neurons, suggesting isoform-specific functions,
it has been proposed that during neurite outgrowth CLASP1 and -2 act in a
redundant fashion by modulating MT dynamics downstream of glycogen synthase
kinase 3 (GSK3). Here, we show that in differentiating N1E-115 neuroblastoma cells
CLASP1 and CLASP2 differ in their accumulation at MT plus-ends and display
different sensitivity to GSK3-mediated phosphorylation, and hence regulation. More
specifically, western blot (WB) analysis suggests that pharmacological inhibition of
GSK3 affects CLASP2 but not CLASP1 phosphorylation and fluorescence-based
microscopy data show that GSK3 inhibition leads to an increase in the number of
CLASP2-decorated MT ends, as well as to increased CLASP2 staining of individual MT
ends, whereas a reduction in the number of CLASP1-decorated ends is observed. Thus,
in N1E-115 cells CLASP2 appears to be a prominent target of GSK3 while CLASP1 is
less sensitive. Surprisingly, knockdown of either CLASP causes phosphorylation of
GSK3, pointing to the existence of feedback loops between CLASPs and GSK3. In
addition, CLASP2 depletion also leads to the activation of protein kinase C (PKC).
We found that these differences correlate with opposite functions of CLASP1 and
CLASP2 during neuronal differentiation, i.e., CLASP1 stimulates neurite extension,
whereas CLASP2 inhibits it. Consistent with knockdown results in N1E-115 cells, primary
Clasp2 knockout (KO) neurons exhibit early accelerated neurite and axon outgrowth,
showing longer axons than control neurons. We propose a model in which neurite
outgrowth is fine-tuned by differentially posttranslationally modified isoforms of CLASPs
acting at distinct intracellular locations, thereby targeting MT stabilizing activities of the
CLASPs and controlling feedback signaling towards upstream kinases. In summary,
our findings provide new insight into the roles of neuronal CLASPs, which emerge as
regulators acting in different signaling pathways and locally modulating MT behavior
during neurite/axon outgrowth