Laboratory of Cell Biology

Partner Group
of the Max PIanck Institute of Molecular Cell Biology and Genetics in Dresden
(the MPI-CBG/IIMCB Partner Group)

Head: Post-doctoral fellows: Research assistant: PhD students: Trainees & undergraduate students:

Marta Miaczynska, PhD HHMI International Research Scholar Wellcome Trust Senior Research Fellow Iwona Pilecka, PhD Beata Pyrzyńska, PhD Rashid Sajid, PhD Magdalena Banach-Orlowska, PhD Marta Brewinska, MSc Anna Zarebska, MSc Anna Hupalowska, MSc Lukasz Sadowski, MSc Anna Torun, MSc Michal Mlacki

RESEARCH

The unifying theme of our research is the relationship between the processes of intracellular membrane transport and signal transduction in response to extracellular stimuli. We would like to understand the molecular mechanisms by which endosomal compartments and their resident proteins play an active role in transmitting intracellular signals. To address these questions we use cultured mammalian cells as models and employ a variety of biochemical and microscopy methods, as well as cell-based functional assays.An increasing number of studies, including our own, indicate that intracellular compartmentalization of signal transduction processes may play an important role in modulating the overall cellular response. It is well established that signals initiated at the plasma membrane, e.g. by binding of ligands to their receptors, are transmitted to the nucleus through the cytoplasm via a series of protein-protein interactions. At first, endocytosis was viewed merely as a mechanism for signal termination by downregulation of surface receptors and their degradation. However, more recent data strongly argue that endosomal compartments and their resident proteins play an important role in transmitting intracellular signals by transporting ligand-receptor complexes and affecting their activity inside the cell. Moreover, the relaying of signals from the plasma membrane via endosomes to the nucleus requires signal mediators to be transported between different cellular locations. Again, a growing number of clathrin adaptors and endosomal proteins are reported to undergo nucleocytoplasmic shuttling. This process is often based on intrinsic sequence motifs and requires active transport mechanisms. Endocytic proteins can associate with nuclear molecules, changing their localization and activity, and may modulate the levels and specificity of gene transcription. As a consequence, certain endocytic genes affect cell proliferation, act as tumour suppressors or change their expression in human cancers. We made an attempt to summarize current knowledge of nuclear functions of endocytic proteins in a recent review (Pilecka et al., 2007). Our previous studies characterising endosomal APPL proteins as signal transducers provided a striking example of the involvement of endosomes in signalling (Miaczynska et al., 2004). Two homologous proteins APPL1 and APPL2 are effectors of the small GTPase Rab5, a key regulator in the early steps of endocytosis. They are localized to a subpopulation of Rab5-positive endosomes that appear segregated from the well-characterized canonical early endosomes marked by another Rab5 effector EEA1. This raised a possibility that APPL-harbouring endosomes may represent a specialized endosomal compartment, potentially involved in signalling. Interestingly, APPL proteins can undergo nucleocytoplasmic shuttling and interact with nuclear proteins, among them the histone deacetylase and chromatin remodelling complex NuRD/MeCP1. Knockdown of APPL1/APPL2 proteins by RNAi demonstrated that each of them is required for efficient cell proliferation. By identifying an endocytosis regulator Rab5 and a nuclear chromatin remodelling complex NuRD/MeCP1 as interacting partners of both APPL proteins, these data pointed for the first time to a direct molecular link between the processes of endocytosis and chromatin remodelling. As histone deacetylase activities are essential for cell cycle progression, APPL binding to NuRD/MeCP1 may serve the purpose of subjecting this function to regulation by extracellular signalling. Moreover, APPL-harbouring endosomes appear as an intermediate in signalling between the plasma membrane and the nucleus. Identification of a novel APPL-mediated trafficking and signalling pathway posed a number of novel questions. The research in our Laboratory currently focuses on the following projects:

Biochemical and microscopical characteriza-tion of an endosomal compartment occupied by APPL proteins.
We apply cell fractionation and gradient purification tech-niques to separate various populations of endosomes in order to enrich APPL-harbouring compartments and determine their protein content. In a parallel approach, we use confocal microscopy to characterize the properties of APPL endosomes and the transport pathways leading through them, in comparison with the canonical early endosomes labelled by EEA1. The quantitative analyses of confocal images are performed in collaboration with Drs. Yannis Kalaidzidis and Marino Zerial (MPI Dresden).To this end, APPL endosomes were visualized by antibody staining and their morphometric features, such as average size of vesicles, their number and fluorescence intensity, were quantified using vesicle tracking algorithms developed by Dr. Kalaidzidis. By internalizing fluorescent cargo (transferrin destined for recycling, epidermal growth factor (EGF) destined for degradation) into HeLa cells using a pulse-chase protocol, a pattern of cargo transport through APPL endosomes was established. These studies demonstrate that both transferrin and EGF are trafficked through APPL and EEA1 compartments but with different kinetics. We concluded that APPL-positive membranes represent a distinct and stable subpopulation of early endosomes, receiving cargo directly from the plasma membrane and capable of differential cargo sorting towards the canonical early and recycling endosomes (Fig. 1). At the moment, we are extending these observations to determine the properties of APPL endosomes, such as their motility and the frequency of fusion events with other endosomal compartments, in living cells. This will be achieved by recording dynamics of APPL endosomes, in comparison with other endosomal markers or cargo, in HeLa cells. Cumulatively, these biochemical and microscopical studies should uncover both the molecular identity and the function of APPL-containing endosomes in trafficking of various cargo molecules.

The mechanisms responsible for APPL1 shuttling in the cell.
We would like to understand the exact roles played by various intracellular pools of APPL1 (endosomal, cytoplasmic and nuclear) and we are searching for compartment-specific determinants localising APPL1 to various organelles. We have determined that APPL proteins undergo a number of posttranslational modifications and we are currently clarifying whether differential modifications could be responsible for the partitioning of APPL proteins into various pools. We would further like to understand whether these pools are interchangeable and which of them is related to APPL function in the regulation of cell proliferation.

The significance of signalling from endo-somes to the nucleus via APPL proteins.
We are characterizing the interactions between APPL proteins and their nuclear binding partners by biochemical methods (co-immunoprecipitation, GST pulldown). In particular, we are interested in the interaction of APPL proteins with the histone deacetylase and chromatin remodelling complex NuRD. This is a multiprotein complex involved in a number of processes, among them transcriptional silencing. We have confirmed that histone deacetylase (HDAC) activity of class I/II can be detected in the immunoprecipitates of APPL1 (Fig. 2). We are also clarifying the intracellular topology of these interactions by analyzing the distribution and trafficking of APPL1 and its nuclear interacting partners by microscopy techniques. We expect that, in the long term, such studies will help to understand how intracellular compartmentalization affects the signalling processes and how molecular communication between endosomes and the nucleus is achieved.

The importance of endocytic transport, including the APPL pathway, in signalling downstream of other growth factors besides EGF.
This task is undertaken in collaboration with other laboratories participating in a European Union Integrated Project entitled: Tracking the Endocytic Routes of Polypeptide Growth Factor Receptor Complexes and their Modulatory Role on Signalling (acronym EndoTrack), in particular with the group of Prof. Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). To this end, we have established methods for detecting the internalized platelet-derived growth factor (PDGF), both biochemically in cell extracts and under the microscope. A biochemical method is based on the quantification of internalized biotinylated PDGF, captured on a streptavidine-coated electrode (Fig. 3). Subsequent detection is performed employing the primary anti-PDGF antibody and a secondary antibody coupled with a ruthenium compound, capable of emitting light when electrochemically stimulated. The light emission is quantitatively measured using an electrochemiluminescence plate reader. The assay was established together with Dr. Patrick Keller from Meso Scale Discovery. We will employ this assay along with the microscopy-based colocalization analyses to measure the amount of endocytosed PDGF in cells under various conditions and to delineate its trafficking pathways. The ultimate goal is to establish whether and by which mechanisms the signal transduction downstream of PDGF depends on its endocytic trafficking.

CV of the Lab Leader

MARTA MIACZYNSKA, PhD
Howard Hughes Medical Institute International Research Scholar
Wellcome Trust Senior Research Fellow

Degrees

• 1997 PhD in genetics, University of Vienna, Austria
• 1992 MSc in molecular biology, Jagiellonian University in Cracow, Poland
• 1991 BSc in biological sciences, University of Wolverhampton, UK

Research Training

• 2001-2005 senior postdoctoral fellow in the Max Planck Institute for Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany
• 1997-2000 postdoctoral training at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany
• 1993-1996 PhD studies in the Institute of Microbiology and Genetics, University of Vienna, Austria
• 1990-1991 exchange student at the University of Wolverhampton, Wolverhampton, UK

Fellowships and awards

• 2007: Habilitation Fellowship of L'Oreal Poland for Women in Science

• 2005: International Research Scholar of Howard Hughes Medical Institute (USA), awarded for 5 years (2006-2010)

• 2005: International Senior Research Fellowship of the Wellcome Trust (UK), awarded for 5 years (2006-2010)

• 2005: Partner Group grant from the German Max Planck Society, (2006-2008)

• 2001-2004 senior postdoctoral fellowship of the Max Planck Society

• 1999-2000 Long Term Postdoctoral Fellowship of the Human Frontier Science Program
  Organization (HFSPO)
• 1998-1999 Erwin Schrödinger Postdoctoral Fellowship from the Austrian Science Fund (FWF)
• 1993-1996 Bertha von Suttner PhD Scholarship from the Austrian Ministry of Science
• 1990-1991 Studentship of the European Community Tempus Scheme

Publications: