Joint research of the Dept.
of Cell Biology is aimed at understanding the pathobiological significance
of (1) cellular energy (ATP) and redox (NAD/NADH and NADP/NADPH) reactions
in cell fate and growth regulation, (2) coupling between energy and
redox metabolism and actin-based cell shape dynamics and cell motility,
and (3) the interdependence between these cellular processes and reversible
protein phosphorylation reactions.
- Next to the transcriptome
and proteome, the "metabolome" forms the third level of
organisation of the cell. Among the >5000 small organic and inorganic
compounds that are present in mammalian cells, energy and redox
metabolites like ATP, phosphocreatine (PCr) and NADH/NADPH have
a very special position. ATP, PCr and NADH/NADPH are synthesized
or utilized in the core pathways of metabolism (glycolysis, PPP-pathway
and TCA-cycle, OXPHOS in mitochondria) and involved in virtually
all interactions between micro- and macromolecular components of
the cell. The importance of energy and redox control for regulation
of cell growth, maintenance of viability, and stress response, which
is directly related to major health problems, is now well documented.
In the majority of reactions involved, ATP and NADH/NADPH and related
pyridine metabolites are used as fuel, electron donors or cofactors.
Past successful efforts of our group have gone into the study of
how phosphotransfer reactions and the distribution of phosphometabolites
are mediated by Creatine Kinases (CK) and Adenylate Kinases (AK).
Our studies were among the first to demonstrate how activities of
these enzymes are coupled to muscle performance and brain activities,
including stress resistance, behavior and learning control. Currently,
we are studying how the homeostasis and dynamic compartmentalization
of ATP and NADH/NADPH is coupled to tumor progression and neurodegenerative
disease.
- Distinctly different
processes like (i) the formation of projections by astrocytes and
neurons, (ii) the development of podosomes and invadopodia by invasive
or malignant cells, (iii) the formation of phagocytic cups by macrophages
or the generation of lamellipodial/filopodial extensions by migrating
or invasive cells, and (iv) even cell division in cytokinesis, share
the hallmark that they are actin-based. These processes also have
in common that they are under temporal and spatial control of ATP
and redox metabolites for the dynamic coordination of actin polymerization
behavior, force generation by myosin ATPases, and local control
of the phosphorylation level of proteins in multiprotein complexes
that steer activity in the cellular cytoskeleton. Currently we are
studying how metabolic enzymes, and thus the local availability
of ATP or redox metabolites, control phagocytosis in macrophages,
osteoclast activity in bone development and osteoporosis, or directional
cell motility in fibroblasts and human glioblastoma cancer cells.
- Metabolic handling
of energy and redox components, of great importance for cell growth,
motility and viability control, is in turn reciprocally coupled
to cell signaling processes that involve the reversible phosphorylation
of serine, threonine or tyrosine residues in cell cortical proteins.
Central in our interest is how activity of members of the protein
tyrosine phosphatase (PTP) family of enzymes and the Rho kinase
family member Myotonic Dystrophy Kinase (DMPK) couples to phosphorylation
of actin-based machinery or energy/redox enzymes in the cell cortex
and determines the fate of neural and muscle cells, epithelial cells
and tumour cells. PTP studies are relevant for a better understanding
of cancer cell behavior, neural development and axon regeneration.
Our work - within an international consortium - originally revealed
that myotonic dystrophy type 1 (DM1), a devastating inheritable
neurodegenerative disorder, is caused by abnormal expansion of a
(CTG·CAG)n repeat in the DMPK gene. New studies now aim to
reveal how cell stress caused by RNA products of this gene cause
muscle and brain problems in patients, and how we can therapeutically
stop this process.
Multidisciplinary Approaches
Our main emphasis in the experimental work is use of state-of-the-art
reverse genetics, molecular cell biology and microscopy in the study
of cellular partitioning behaviour and biological and pathobiological
role of our pet energy/redox/signaling enzymes.
To provide an adequate description of the molecular environment of
the molecules of interest we use partner-protein identification methods
like yeast two-hybrid screening, co-immunoprecipitation and protein-complex
pull-down, 2D gelelectrophoresis and mass-spectrometric protein identification.
New bioinformatic approaches like data mining and prediction of protein-structure/interaction
are currently being introduced in collaboration with the CMBI
department. Full-length proteins or segments are produced in pro-
and eukaryotic host systems, and scanning mutagenesis and 3D structure
determination is exploited to obtain a better understanding of the
structural requirements for complex assembly and routing towards distinct
cell compartments. In the past, large efforts have gone in the characterization
of the properties of catalytic domains in amphiphatic proteins like
CK and AK, the PDZ domains in membrane-bound PTPs like PTP-BL, or
the mitochondrial and ER-binding role of C-terminal tail anchors in
DMPK isoforms.
Another major challenge is to collect data on the variation in subcellular
distribution and intracellular flux direction of small, soluble metabolites
(ATP, PCr, ions) and the role of the cytoarchitectural organization
in cellular energetics and differentiation. To study these aspects,
we use sophisticated microscopical procedures, both at the light-microscopical
level (CSLM, video microscopy) and ultrastructural level (immuno-EM,
SEM, AFM). Protein behavior is studied by use of various tagging procedures
(Green Fluorescent Protein derivatives, Myc/VSV/FLAG-tags) and for
recording of metabolite-ion behavior we use fluororescent or FRET-based
reporters and real-time imaging methodology.
Finally, to gain insight in the pathophysiological significance of
our systems transgenic and knockout mouse models are available to
study the consequences of mutations or ectopic expression at the organismal
level. Importantly, the animal models also serve as source for the
isolation of embryonic fibroblasts, immortalized myoblasts-myotubes
or brain cells, enabling us to do cell biological studies and go one
step back in complexity. Alternatively, siRNA knockdown procedures
are being used for the generation of cell models. Physiological or
behavioral testing is performed to monitor gene function. At the cell
level transient or permanent DNA transfection-complementation with
full-length or domain-specific cDNA constructs or viral transduction
(with adenoviral, retroviral or baculoviral vectors) are used to monitor
gene-function relationships. Through collaborations with the Departments
of Radiodiagnostics
(Heerschap, UMC St.Radboud), Biophysica_newl
Chemistry (Vuister) and Tumor
Immunology (Figdor, NCMLS) the above methods are complemented
with physical approaches like in vivo Nuclear Magnetic Resonance Imaging
(MRI) and Spectroscopy (NMS), protein domain structure determination
via NMR, the study of binding kinetics using surface plasmon resonance
(SPR), and the use of scanning probe/atomic force microscopy (AFM)
and fluorescence microscopy.
Internship/Traineeship Information for MSc students and Ph.D. fellows
We are located on the sixth floor of the NCMLS Research Building,
where also most of the instruments and facilities of the Microscopic
Imaging Center are localized (see http://www.ncmls.nl/celbio/pandf.htm).
Our department has weekly progress report meetings and literature
seminars, in which scientists, technicians and students participate.
Msc students and Ph.D. students are supposed to also attend lectures
that are scheduled in the educational program of the NCMLS. MSc students
are assigned to one specific research project for the entire duration
of their training period. The subject of study is dependent on the
current status of the ongoing investigations and is determined - after
taking into account the candidate's preferences - just prior to the
actual commencement of the traineeship period. Experienced PhD students
or post-doc scientists working on the pertinent research topic together
with group leaders (Fransen, Hendriks,
Wieringa) provide supervision. The working environment
at the department is informal but dedicated and we expect students
to be actively engaged in all departmental activities. The theoretical
component of the MSc study entails (i) writing of a full master's
thesis report about the trainee period, (ii) an oral examination based
on chapters from one current molecular cell biology textbook (e.g.,
Alberts et al., Molecular Biology of the Cell, 4th edition; Garland
Press) and (iii) writing of a short review on a newly discovered cell
biological concept (title to be established). Ph.D. studies will be
embedded in the NCMLS Ph.D. program (see www.ncmls.ru.nl).
Suitable profile:
All MSc students with a chemical/biological, biological/physical or
biomedical profile, with molecular biological and/or cell biological
elements in their training program are invited to apply for internship
training opportunities. An experienced supervisor is assigned to each
individual student. Hence, we have only a limited number of student
positions available at a given moment during the academic year. Interested
Ph.D. students can apply for open vacancies listed on the department
or NCMLS web sites.
|
