LCAM Internships

 

LCAM is always interested in motivated masterstudents that want to do an internship in one of the three labs. Below a summary is given of the various projects running. However, because of fast developments in research it’s not always possible to maintain an actual list of all ongoing projects. Therefore more information on projects, related to the scientific research of the specific LCAM lab, can be retrieved from the principal investigators.

Internships in LCAM-FNWI, -AMC and -NKI:

Guiding cell movement through modifying cytoskeletal signaling networks using optogenetics
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular Cytology
Examinator: dr. ir. Joachim Goedhart
Supervisor: Orry van Geel

Description:
Controlling the cytoskeleton involves many signaling pathways interacting with each other, but we want to identify a minimal set of components that can affect microtubules on their own. The eventual goal is then to use these components in an in vitro system to create synthetic vesicles that can change shape or migrate. First however we must identify these components in cells by studying the effects of different signaling proteins on microtubules. This project will focus on identifying proteins that have big impacts on microtubule dynamics using optogenetic tools to translocate proteins or to switch protein activity on or off.

Technical skills/methods:
During this project you will perform mammalian cell culture and molecular cloning to make new optogenetic fusion constructs with signaling proteins and test them on confocal microscopes. Readouts can be acquired by different advanced imaging techniques like FRET, FLIM, and FRAP.

Timespans: >36 EC = >24 weeks

Contact: Orry van Geel, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Labelling & Imaging Strategies for Prolonged Imaging of Mycobacteria
Research organization: LCAM-AMC, Academic Medical Centre
Institute: AMC, Department of Cell Biology and Histology
Examinator: Dr. Nicole van der Wel
Supervisor: Dr. R.A. Hoebe & Venkat Krishnaswami

Description:
The study of DNA in Mycobacteria using fluorescence imaging can be impaired by the effects of photodamage via photobleaching and phototoxicity. Several factors ranging from labelling methods to imaging modes and techniques can be responsible for photodamage. The aim of this project is to study the effects of photodamage, improve the labelling strategies for Mycobacteria and image them by employing different imaging modes and techniques, including the state-of-the-art confocal & widefield microscopes. Additionally, imaging of Mycobacteria could be performed using a novel microscopic technique that has been developed in our lab, especially to reduce the effects of photodamage. The effects of photodamage in Mycobacteria using different labelling dyes and imaging techniques is performed by basic image analysis using tools like ImageJ and/or Matlab.              

Techniques:
For this project the student is expected to perform cell & bacterial-culturing and imaging using different advanced microscopic techniques, perform basic image analysis using Imagej/Matlab etc.

Timespan: >36 EC = 24 weeks

Contact: R.A. Hoebe (r.a.hoebe@amc.uva.nl) or Venkat Krishnaswami (v.krishnaswami@amc.uva.nl), Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam.

 

Development of cAMP sensors
Research organisation: LCAM-NKI, Netherlands Cancer Institute
Department: Jalink lab
Examinator: Dr. Kees Jalink (NKI)
Supervisor: Dr. Kees Jalink (NKI)

Description: Our group has a long history in developing biologically relevant FRET sensors, to measure for instance Estrogen Receptor functioning, phosphatidyl inositol bisphosphate (PIP2) and cyclic AdenosineMonoPhosphate (cAMP). cAMP is an important second messenger that activates Protein Kinase A (PKA). Our lab recently published a new EPAC-based sensor to measure cAMP (Klarenbeek et al PlosOne 2011). However further improvements to this sensor can still be made in both sensitivity and stability of the sensor during long measurements. Another way of detecting cAMP is by using a PKA-based sensor. PKA is more sensitive to cAMP than EPAC, but has the disadvantage that expression of the sensor often is troublesome. New molecular tools have to be developed to allow reliable expression of the PKA-based sensor. All new sensors have to be tested and validated using advanced microscopic techniques. In line with the cAMP sensor we are also interested in (local) induced effects of cAMP by adenylate cyclase. Recently a Photoactivatable Adenylate Cylcase (PAC) is published. This PAC is able to activate adenylate cyclase in distinct parts of the cell by activating it by laser light. This method allows us to test the effects of cAMP on distinct structures of a cell. Also other photoactivatable receptors are currently made or have been published recently, for instance photoactivatable G-Coupled receptors (paGPCR’s). By this method we are able to activate by laser light. Some paGPCR’s have been published, but others are still under construction and have to be fully characterized.

Technical skills/methods: These developments constitute ideal student projects. In the course of ~8 months, you will learn about molecular cloning, fluorescent proteins, FRET and the instrumentation you need for that. You will create new, improved version(s) and extensively test them with state-of-the-art equipment. Depending on the progress made, you will also be able to apply your own FRET sensor to address questions embedded in one of the research lines in our lab.

Timespans: >60 EC = 42 weeks

Remarks: Because of the speed of developments, it is inpossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.

Contact: Kees Jalink,  tel.: 31-20-512-1933/1947
NKI-AVL Plesmanlaan 121 Amsterdam

 

Imaging glial cells in Huntington’s Disease
Research organization: LCAM-AMC, Academic Medical Centre
Institute: AMC, Protein Degradation and Aggregation,
Department: Department of Cell Biology and Histology
Examinator: Anne Jansen (AMC)
Supervisor: Dr. Eric Reits (AMC)

Description: Huntington’s disease (HD) is a neurodegenerative disease caused by accumulation and aggregation of polyglutamine (polyQ)-expanded Huntingtin (htt) protein fragments. While the expanded polyQ Huntingtin protein is expressed in all cells, most researchers assume that the disease is primarily caused by the polyQ-expanded proteins expressed in neurons since these cells are most vulnerable. However, the majority of the cells in the central nervous system are nonneuronal glial cells such as astrocytes and microglia, which express htt as well. Glial cell dysfunction will directly affect neuron-glial interactions and synaptic functioning, which emerges as an important factor in HD pathogenesis.

We hypothesize that glial cells play an essential role the development and progression of HD. Our project will consists of two parts:

  • Visualize and compare htt aggregation in glial cells versus neurons in brain tissue of HD mouse models. Preliminary data suggest that astrocytes and microglia do contain huntingtin aggregates, but that these aggregates are smaller and might have a different location in the cell when compared to neurons. These differences might explain why glial cells are better able to handle the polyQ proteins. Aggregates will be visualized by electron and confocal microscopy.

 

  • Examine differences in protein degradation machineries. Glial cells      might be better in cleaning up htt aggregates due to a differently working protein degradation system (e.g. the ubiquitin proteasome system and autophagy). Using confocal microscopy and biochemistry, we want to visualize proteasome levels and activity in neuronal cells and glia. For  this approach we will use different activity probes and antibodies for the ubiquitin proteasome system components, Huntington and different cell types.

Techniques:
Electron and confocal microscopy,
Immunostaining,
Westernblotting

Timespan: >60 EC = 42 weeks

Contact: Anne Jansen (a.h.jansen@amc.uva.nl) and Eric Reits (e.a.reits@amc.uva.nl)
Protein Degradation and Aggregation, Department of Cell Biology and Histology
Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

 

Partner or social dance?-Quantification of MAPK protein complexes
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular Cytology
Examinator: dr. ir. Mark Hink
Supervisor: dr. ir. Mark Hink

Description:
Mitogen-activated protein (MAP) kinase pathways play a very important role in cell functions such as cell proliferation, differentiation, survival, and tumorigenesis and misfunction of these pathways lead to severe damage. One of the intriguing questions is that a different isoform composition of the complex of the involved proteins (Ras, Raf, ERK, MEK & KSR) can lead to a different output signal. The aim of this project is to quantify, by using confocal microscopy in combination with advanced fluorescence spectroscopy (f.e. FCCS), the interaction of these MAPK signaling complexes in the living cell and study the effect of pathway stimulation and protein isoforms.

Technical skills/methods: Dependent on the length of the project and the interest of the student, one has the possibility to work on several different disciplines, including molecular biological (cloning), cell culturing, advanced fluorescence microscopy (FRET-FLIM, FCCS and ICCS) and data analysis.

Timespans: >36 EC = >24 weeks

Contact: Mark Hink, Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

EGFR internalization by ALPs
Research organisation: LCAM-NKI, Netherlands Cancer Institute
Department: Cell Biology
Examinator: dr. Kees Jalink
Supervisor: dr.  Kees Jalink

Description:
The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor whose signaling modulates cell growth, differentiation, survival, adhesion and migration. The dysregulation of EGFR signaling leads to tumorigenesis. EGFR is found both overexpressed and mutated in a variety of human cancers. Several anti‐EGFR therapies using monoclonal antibodies and tyrosine kinase inhibitors have been developed, and are either in use or in clinical trials. We are studying the possibility of targeting EGFR with ether‐linked (alkyl) lysophospholipids (ALPs). We are analyzing how these may induce EGFR internalization. For that, we need to quantify internalization in a dose-dependent manner, identify the pre-requisits and mechanism for it and finally see it in living cells. This will be attempted by immuno-fluorescence microscopy using specific antibodies, and a specific EGFR-tag. We seek an enthusiastic student to fill in this ‘gap’ in our data, willing to learn cell culture, transfections, live-cell imaging, immuno-fluorescence and cloning. Because of the speed of developments, it is inpossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.

Technical skills/methods: These developments constitute ideal student projects. In the course of ~8 months, you will learn about molecular cloning, fluorescent proteins, FRET and the instrumentation you need for that. You will create new, improved version(s) and extensively test them with state-of-the-art equipment. Depending on the progress made, you will also be able to apply your own FRET sensor to address questions embedded in one of the research lines in our lab.

Timespans: >60 EC = 42 weeks

Remarks: Because of the speed of developments, it is inpossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.

Contact: Kees Jalink,  tel.: 31-20-512-1933/1947 NKI-AVL Plesmanlaan 121 Amsterdam

 

Effects of lipids and anti-cancer drugs on PKC
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular Cytology
Examinator: dr. ir. Joachim Goedhart
Supervisor: dr. ir. Joachim Goedhart

Description:
Protein kinase C (PKC) plays a key role in signal transduction cascades that involve phospholipid hydrolysis and has been implicated in many processes including proliferation, differentiation, carcinogenesis and apoptosis. The PKC
family is divided in three classes; classical, novel and atypical. Both the classical and novel PKC isoforms have C1-domains that can bind an important signaling lipid; diacylglycerol (DAG). The C1-domains recruit PKC to membranes where DAG is formed, thereby activating its kinase activity. At this moment, several drugs that target C1 domains are currently studied as potential
anti-cancer drugs in clinical trials. So far binding studies with PKC and isolated C1-domains have mainly been done in vitro. To  obtain a better understanding of how the drugs and natural lipids act on PKC and the C1-domains we study this process in the relevant environment, i.e. the living cell. To this end, green fluorescent protein (GFP) is fused to these proteins and the
constructs are expressed in cells. The (trans)location of GFP-tagged proteins is studied by fluorescence microscopy in real time with high spatial resolution. Cutting-edge microscopy methods are used to follow multiple proteins in a single cell by using spectrally different fluorescent proteins. This approach allows to study in detail the mechanism by which lipids and drugs
bind to PKC in living cells, shedding light on the role of PKC in signaling and cancer.

Technical skills/methods: For this research a variety of techniques will be used including, molecular biology (cloning), eukaryotic cell culture and(advanced) fluorescence microscopy (FRET, FRAP, TIRF).

T
imespans: >30 EC = >20 weeks

Contact: Joachim Goedhart, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

 

Visualize the invisible- Image correlation analysis of G protein signaling
Research organisation: LCAM-FNWI, University of Amsterdam
Institute: SILS
Department: section of Molecular Cytology
Examinator: dr. ir. Mark Hink
Supervisor: dr. ir. Mark Hink

Description:
We want to quantify the concentration, mobility and degree of interaction of G-protein coupled receptors (GPCR) and their downstream signalling components in living cells. Thereto more sensitive and selective techniques are needed. Fluorescence fluctuation spectroscopy (FFS) methods are promising tools since they can detect fluorescently labeled molecules down to the single-molecule level, even in the living cell. The aim of this project is to use the available (high speed) confocal microscopes in combination with sensitive detectors in order to test, optimize and apply recently developed FFS techniques as RICS, STICS and kICS (Kolin et al., Cell Biochem. Biophys. 49: 141 (2007)) in order to study the fluorescently labeled proteins in living HeLa and HEK cells.

Technical skills/methods:
Dependent on the length of the project and the interest of the student, one has the possibility to work on several different disciplines, including cell culturing, advanced fluorescence microscopy (FLIM, FCS and ICS) and data analysis (development).

Timespans
: >30 EC = >20 weeks

Contact: Mark Hink, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

 

Epigenetic manipulation of Wnt expression in the mammary gland
Research organization:  LCAM-FNWI, University of Amsterdam
Institute: SILS
Department: section of Molecular Cytology
Examinator: dr. Renée van Amerongen
Supervisor: Katrin Wiese & Renee van Amerongen

Description: Wnt ligands play a crucial role during embryogenesis and adult tissue homeostasis, controlling processes such as self-renewal, proliferation and cell fate determination. Conversely, aberrant Wnt signaling contributes to a variety of human diseases, most notably cancer. Hence, Wnt pathway activity has to be tightly regulated in specific spatio-temporal patterns but we are still lacking a clear understanding of the underlying control mechanisms.

DNA regulatory sequences called enhancers are important determinants of distinct gene expression patterns in multicellular organisms. Therefore, identification of these functional elements will be an important step towards deciphering the complex regulatory circuits that control Wnt expression. However, enhancers often influence gene expression from a considerable distance and their identification in vivo is a challenge.

The specific goal of this MSc project is to establish a robust experimental system that will allow high-throughput screening of putative enhancer sequences. Using this approach, the functionality of candidate enhancers to drive Wnt expression can be rapidly and thoroughly tested before characterizing them further in in vivo transplantation assays.

To this end, you will use CRISPR genome editing technology to manipulate the transcriptional control of endogenous Wnt ligands in murine mammary epithelial cell lines.

Experimental approach

  • Generate a panel of fluorescent reporter cell lines to monitor expression of different Wnt proteins.
  • Generate a clonal cell population expressing a dCas9-p300 fusion protein that will allow you to target and transcriptionally activate promoters and enhancers of your choice.
  • Design guideRNAs to target dCas9-p300 to candidate regulatory sequences.
  • Verify alterations of epigenetic marks at these sites by chromatin-immunoprecipitation (ChIP).
  • Monitor modulation of Wnt expression by qRT-PCR, FACS and confocal microscopy.

Technical skills / methods: Molecular cloning, CRISPR, lentivirus production, (primary) cell culture, fluorescence-activated cell sorting (FACS), confocal microscopy, qRT-PCR and chromatin immunoprecipitation (ChIP).

Timespans: >36 EC = >24 weeks 

Contact: Renée van Amerongen, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

G-protein coupled receptor (GPCR) signaling unraveled
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular Cytology
Examinator: dr. ir. Joachim Goedhart
Supervisor: Marieke Mastop

Description:
GPCR signaling is involved in many important cellular processes and is a popular drug target; over 30% of all known drugs act through GPCRs. More than 60% of the membrane receptors are GPCRs. GPCRs activate heterotrimeric G-proteins , consisting of an α, β and γ subunit. There are only 4 different subfamilies of Gα-proteins that are activated through GPCRs: Gαq, Gαi, Gαs and Gα12/Gα13.

It is still not clear how specificity in GPCR signaling is achieved. The classic model in which one GPCR activates one specific isoform of the four Gα classes has changed in the last decade. It is becoming clear that GPCRs can exist in multiple states, each with a certain affinity for a specific G-protein.

Technical skills/methods:

Timespans: >30 EC = >20 weeks

Contact: Marieke Mastop, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Optimizing fluorescent probes to develop a next generation highly sensitive FRET based biosensors
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular Cytology
Examinator: dr. ir. Joachim Goedhart
Supervisor: Marieke Mastop

Description:
GPCR signaling is involved in many important cellular processes and is a popular drug target. More than 60% of the membrane receptors are GPCRs and over 30% of all prescribed drugs act through GPCRs. GPCRs activate heterotrimeric G-proteins, consisting of an α, β and γ subunit. There are 4 different subfamilies of Gα-proteins that are activated through GPCRs: Gαq, Gαi, Gαs and Gα12/Gα13.

It is still not clear how specificity in GPCR signaling is achieved. The classic model in which one GPCR activates one specific isoform of the four Gα classes has changed in the last decade. It is becoming clear that GPCRs can exist in multiple states, each with a certain affinity for a specific G-protein family. In our group FRET based biosensors are developed to measure the activation of Gαq and Gαi. Additionally, a biosensor for Gα13 is in progress.

This project involves functionally tagging Gαs with fluorescent proteins to make a biosensor for this Gα class as well.

Technical skills/methods:
Because free N- and C- termini of the Gα subunit are essential for functionality, the fluorescent protein has to be inserted within the protein sequence. Subsequently, the functionality of the fusion has to be determined and after successful insertion, the variant can be used to generate a biosensor

This project involves molecular biology techniques like cloning and cell culture as well as advanced imaging techniques as FRET, FLIM and FCS.

 Timespan: >30 EC = >20 weeks

 Contact: Marieke Mastop, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Endogenous RhoGTPase signaling in vascular endothelial cells
Research organisation: LCAM-FNWI, University of Amsterdam & Sanquin
Department: section of Molecular cytology/Molecular Cell Biology
Examinator: Prof. dr. T.W.J. Gadella
Supervisor: Nathalie Reinhard

Description:
RhoGTPases are crucial regulators of the actin cytoskeleton, thereby controlling cell adhesion and migration. They participate in a broad spectrum of fundamental biological processes related to human health and disease, including cancer cell migration, invasion, and metastasis, inflammation, and wound repair. In this study we specifically focus on RhoGTPases in endothelial cells in which they are linked to vascular integrity. Although these proteins have been studied for many years in this cell system, previous studies were all based on overexpression of these proteins. The aim of this project is to study RhoGTPases on an endogenous level. This might give us new spatiotemporal information and allows us to study physiological relevant signaling pathways in great detail.

Technical skills/methods: During this project a variety of techniques will be combined to unravel RhoGTPase characteristics and signaling pathways, including molecular cloning, CRISPR, mammalian cell culture and several advanced microscopy techniques (confocal microscopy, FRET, FLIM, TIRF).

Timespans: >36 EC = >24 weeks

Contact:
Nathalie Reinhard, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Quantifying endogenous Wnt signalling in real-time
Research organization:  LCAM-FNWI, University of Amsterdam
Institute: SILS
Department: section of Molecular Cytology
Examinator: dr. Renée van Amerongen
Supervisor: Anoeska van de Moosdijk , dr. Renée van Amerongen & dr. Mark Hink

Description: We have successfully set up CRISPR/Cas9 technology in our lab to fluorescently tag a gene named beta-catenin, a key player in the Wnt signalling pathway. Because of its importance for tissue development and homeostasis and its role in stem cells maintenance, this pathway has been well studied for several years.

We still need to gain a far more detailed understanding of the activities in the Wnt pathway in healthy and diseased tissues. Little is known about the exact signalling events happening upon Wnt stimulation. But times are changing: CRISPR/Cas9 genome editing now allows us to tag genes endogenously, opening up possibilities for studying signalling in real time and at real concentrations.

The goal of this project is to use advanced microscopy techniques to measure beta-catenin dynamics in living cells, using a cell line containing endogenously tagged CTNNB1, the gene encoding beta-catenin.

You will use advanced microscopy to further characterize beta-catenin in the Wnt signalling pathway. Depending on your interests, you will

  1. Use CRISPR/Cas9 to tag beta-catenin binding partners (Axin1, Gsk3, TCF/LEF) and perform FCCS experiments to study their dynamics via a multi-colour approach.
  2. Use CRISPR/Cas9 to tag beta-catenin in a mouse mammary gland cell line and compare its dynamics to what we know from the already developed human cell line.
  3. Use CRISPR/Cas9 to create known tumorigenic mutations in the fluorescently tagged CTNNB1 allele and study dynamics of the constitutively active variants.

 Technical skills / methods: CRISPR technology, DNA cloning, cell culture, advanced microscopy (FCS / FCCS, FRAP)

Timespans: >36 EC = >24 weeks
Contact: Renée van Amerongen, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Improving FRET based biosensors that report on signaling of oncogenic players
Research organisation: LCAM-FNWI, University of Amsterdam
Institute: SILS
Department: section of Molecular Cytology
Examinator: dr. ir. Joachim Goedhart
Supervisor: Marieke Mastop

Description: GPCR signaling is involved in many important cellular processes including cell proliferation and cell migration, which are important processes in the development of cancer and metastasis. Mutations and aberrant expression of GPCRs and G proteins have been linked to several type of tumors (head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer, breast, prostate and gastric tumours, melanoma and diffused large B cell lymphoma).

GPCRs activate heterotrimeric G-proteins, consisting of an α, β and γ subunit. There are only 4 different subfamilies of Gα-proteins that are activated through GPCRs: Gαq, Gαi, Gαs and Gα12/Gα13. Each of the Gα subtypes induces distinctive downstream signaling. Downstream effectors of GPCR signaling, such as the Rho family of small GTPases (Rho, Rac, and Cdc42), play a key role in cell migration and are as such linked to metastasis. The details about how only 4 different Gα subunit types can render very specific signaling outcomes is still to be determined. The more knowledge we gather, the more specifically we will be able to interfere with the GPCR signaling in cancer therapeutics.

In our lab, GPCR signaling is studied using FRET based biosensors that can report on the activation of certain biological players in real-time in living cells. These sensors are employed to study for example the activation of Gαq, Gαi or Gα13, RhoA and Rac and they can also report on intracellular levels of for example cAMP or calcium.

Technical skills/methods:

During this project you will focus on 1) improving biosensors to make them more sensitive and to increase their dynamic range, 2) characterization of the novel biosensors and on 3) using improved biosensors to measure multiple signaling processes simultaneously in living cells. This project involves molecular biology techniques like cloning and cell culture as well as advanced imaging techniques as FRET, FLIM and FCS.

Timespan: >30 EC = >20 weeks 

Contact: Marieke Mastop, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

The in vivo mechanism of AnnexinA4 aggregation in mammalian cells
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular Cytology
Examinator: Dr. Marten Postma or dr. ir. Mark Hink
Supervisor: Dr. Marten Postma

Description: Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis, ion-transport and protein kinase C signaling. However, its precise mechanistic role and Ca2+-induced aggregation mechanism is not fully understood. Ca2+-induced aggregation on the intracellular membranes may be critical for its function.

In this project we will study the mobility and self-assembly of AnxA4 after Ca2+ influx at the plasma membrane in living HeLa cells. Existing fluorescent protein fusion proteins and newly created binding mutants affecting aggregation will be studied by advanced microscopy techniques. The resulting images will be analyzed by newly developed analysis software.

Technical skills/methods: Dependent on the length of the project and the interest of the student one has the possibility to work on several different disciplines, including molecular biology, cell culturing, confocal microscopy, advanced fluorescence microscopy (FRET-FLIM, FCS, FRAP and N&B) and data analysis (development). References: Crosby et al., Biophys J. 104:1875-1885 (2013)

Timespans: >30 EC = >20 weeks

Contact: Marten Postma, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Studying RhoGTPase signaling pathways in endothelial cells under flow
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular cytology
Examinator: dr. ir. Joachim Goedhart or prof. dr. T.W.J. Gadella
Supervisor: Nathalie Reinhard

Description: Human endothelial cells line the vasculature and control physiological processes, including regulation of blood pressure, clotting and the transendothelial migration (TEM) of leukocyte and tumour cells. Consequently, endothelial cells play a central role in pathologies such as cancer and inflammation, forming a barrier for the migration of cells from the circulation to the tissues. It has been recognized that the endothelium is not a passive layer of cells but actively participates in the process of leukocyte- and tumour cell extravasation. The molecular mechanisms underlying these processes highly depend on RhoGTPase functioning, a group of proteins involved in the regulation of the cytoskeleton. Although these proteins play a major role in vascular integrity they have been mainly studied under static conditions. The aim of this study is to use a physiological relevant approach to study RhoGTPase signalling in the context of endothelial barrier function.

Technical skills/methods During this project a flow system will be used to mimic the human blood flow. In addition, a variety of techniques will be combined to unravel crucial RhoGTPase signaling pathways, including molecular cloning, biochemical assays, mammalian cell culture and several advanced microscopy techniques (confocal microscopy, FRET, FLIM, TIRF).

Timespans: >30 EC = >20 weeks

Contact: Nathalie Reinhard, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

In vivo localization of M. tuberculosis
Research organisation: LCAM-AMC, University of Amsterdam
Department: section of Molecular cytology
Examinator: dr Nicole van der Wel
Supervisor: Dr. Nicole van der Wel

Description: In the past few years we have shown in cell cultures that the localization of Mycobacterium tuberculosis is crucial for its pathogenicity (van der Wel et al., Cell 2007, Houben et al., Cell Micr 2012). While, vaccine strain M. bovis BCG and other non-pathogenic mycobacteria, remain constrained in the membrane enclosed compartments, all pathogenic mycobacteria are able to escape from their membrane enclosed compartments and float freely in the cytosol (Houben et al., Cell Micr 2012). The correlation between the localization and the pathogenicity was described using macrophage or dendritic cell culture systems which is essential to be able to synchronize the infection and generate comparable data.

As we shown that the escape of M. tuberculosis determines its pathogenicity, we have studied the localization of M. tuberculosis in the lungs of mice (B6,Balb/c). Surprisingly, we detected mostly membrane enclosed M. tuberculosis. Only very early in infection, when the adaptive immunity is not yet present, we detected bacteria that escaped their membranous compartment (Figure 1) similar to what we described for cell culture systems. Preliminary results that the bacteria are able to escape but further analysis at day 2,7,45 and 120 are needed and thus part of the proposal. In this project but fluorescent microscopy as well as electron microscopy will be performed. First FM will be used to determine the site of infection in skin tissues, long tissues of mice or zebra fish embryos. Then the tissue will be analyzed using electron microscopy.

Technical skills/methods
Localise Mycobacteria in already prepared tissues of mice, leprosy patients or zebrafsh using fluorescent microscopy.
Determine the subcellular localisation of Mycobacteria in tissues using electron microscopy.

Timespans: >30 EC = >20 weeks

Contact: Nicole van der Wel, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam

 

Building novel Wnt-pathway reporters
Research organization:  LCAM-FNWI, University of Amsterdam
Institute: SILS
Department: section of Molecular Cytology
Examinator: dr. Renée van Amerongen
Supervisor: Anoeska van de Moosdijk , dr. Renée van Amerongen

Description:
The Wnt signal transduction pathway is essential for embryonic development and tissue maintenance in all multicellular animals. In mammals, it controls cell proliferation and cell fate decisions. This is both a power and an Achilles heel. On the one hand, Wnt signalling can promote stem cell maintenance and aid in tissue repair. It thus holds great promise in the field of regenerative medicine, and could potentially be used for directed differentiation of cells and tissue bio-engineering approaches. On the other hand, Wnt signalling is frequently deregulated in human tumours, making it a useful target for therapeutic intervention.

Axin2 is a negative feedback regulator of the Wnt-pathway. It is often used to monitor Wnt-pathway activation in multiple cells and tissues. In fact, Axin2 is the core of many of the experiments we perform in the lab. We want to build a tool to monitor Axin2 activity in real time in vitro and use this information do develop a novel reporter in vivo.

Since we are always looking for ways to build better tools and mouse models to monitor Wnt signalling, you will use the CRISPR/Cas9 technology to insert fluorescent tags into the Axin2 locus and compare their behaviour in response to Wnt-pathway activation. Inserting tags is done via Homology-Directed Repair (HDR), a bottle-neck in CRISPR/Cas9 directed gene editing. You will use different approaches to increase HDR for the Axin2 locus, such as the use of novel Cas9 variants.

You will use CRISPR/Cas9 technology to insert fluorescent tags into the Axin2 locus and compare their behaviour in response to Wnt-pathway activation. Depending on your interests, you will measure and quantify

  • The use of multiple copies of fluorescent proteins and/or a localization signal (such as NLS) to reach detectable fluorescent signals upon low endogenous expression.
  • Use novel Cas9 variants, such as (inducible) Cas9-Geminin to increase HDR-levels.
  • Test HDR-enhancers to increase HDR-levels.
  • Test micro-homology or asymmetric homology arms to increase HDR-levels.
  • Test the use of single-stranded homology arms to increase HDR-levels.

 

 Technical skills / methods: CRISPR technology, DNA cloning, cell culture, confocal microscopy, Western Blot analysis

Timespans: >36 EC = >24 weeks

Contact: Renée van Amerongen, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

High resolution imaging of M. tuberculosis DNA
Research organisation: LCAM-AMC, University of Amsterdam
Department: section of Molecular cytology
Examinator: dr Nicole van der Wel
Supervisor: Nicole van der Wel

Description: Mycobacteria, like other gram negative bacteria, do not have a nucleus, but they store most of their DNA in specific compartment in the cell that is not delimited by a membrane. This compartment is often referred as nucleoid. It has been observed for E.coli and B. subtilis (Dwyer et al. 2012, Borgmann at al. 2013) that, in response to stress, the genome of the bacteria seems to condensate even further forming big nucleoid. This condensation mechanism is reported to correlate with bacteria-death.

We have found that in Mycobacteria a specific antibiotic induces nucleoid-condensation, while other antibiotics kill mycobacteria without inducing condensation of the DNA. In other words we detected that there are at least two different mechanisms of antibiotic induced cell-death. As in healthy mycobacteria the nucleoid has a diameter of around 200nm and therefore we need to image the nucleiod in intact bacteria. We would like to measure the size of the nuceloid in mycobacteria treated using both fluorescence microscopy (FM) and electron microscopy (EM). The measurement of the change in volume of the nucleoid should correlate with the amount of DNA as detected by DAPI staining. The images should be correlated so the project will involve Correlative Fluorescence Electron Microscopy (CLEM).

Technical skills/methods

  1. Correlative light-electron microscopy (CLEM); image bacteria using EM and correlate to FM.
  2. Study effects of different antibiotic on DNA distribution.
  3. Compare death by starvation, to death by antibiotics.

Timespans: >30 EC = >20 weeks

Contact: Nicole van der Wel, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam

 

 

Trafficking of the novel membrane protein Tmem98
Research organisation: LCAM-FNWI, University of Amsterdam
Department: section of Molecular cytology
Examinator: dr Renee van Amerongen
Supervisor: Renee van Amerongen

Description: A couple of years ago, we performed a yeast-two-hybrid screen to identify novel interaction partners for Frat2, an activator of the Wnt/b-catenin pathway. This resulted in the identification of Tmem98, a novel membrane protein of unknown function. We have performed a thorough biochemical characterization of Tmem98 and found that Frat1/2 and Tmem98 function in a feedback loop, in which they reciprocally affect each other’s protein levels. We also found that Tmem98 traffics from the Golgi to the plasma membrane and is internalized via endocytosis, after which it appears to be recycled.

You will use advanced microscopy to further characterize the dynamic trafficking and signaling behavior of Tmem98.

  1. Our initial experiments point towards Tmem98 being a single-pass transmembrane protein, with the N-terminus serving as a signal peptide and a transmembrane anchor and the C-terminus sticking out into the cytoplasm. You will confirm this orientation by a fluorescence protease protection (FPP) assay.
  2. Our current Tmem98-GFP fusion protein appears to have lost (some of) its signaling function. Therefore, you will generate a novel Tmem98-GFP fusion, in which you will insert the fluorescent protein into an internal loop of the protein. Next, you will repeat some of the earlier trafficking experiments and confirm inibition of Wnt-signaling in response to Tmem98.
  3. You will confirm some of the Tmem98 targets that came out of our previous microarray analyses by qPCR analysis of Tmem98 knockdown or CRISPR/Cas9 knockout cells.
  4. You will perform live-cell confocal microscopy to follow the trafficking of Tmem98 in cells that co-express fluorescently labeled Rab proteins.

 

Technical skills / methods: DNA cloning, cell culture, confocal microscopy

Timespans: >36 EC = >24 weeks

Contact: Renée van Amerongen, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

 

Structure of the Keratin Network at Super-Resolution
Research organisation: LCAM-NKI, Netherlands Cancer Institute
Department: Cell Biology
Examinator: dr. Kees Jalink
Supervisor: dr.  Kees Jalink & Leila Nahidi

Description:
Cells contain 3 different cytoskeleton systems: thin actin filaments, thick microtubuli and also intermediate filaments (IF) such as keratin and vimentin, which have been much less studied. Keratin filaments are major cytoskeletal components of the epithelial cells that cover external and internal surfaces of the human body. Keratin IF fulfill essential roles by providing mechanical integrity to the skin, gut mucosa and other epithelial tissues. They also participate in a wide range of cell functions such as organelle trafficking, motility, translation, signaling, immune response, and cell survival. Keratin malfunctions are therefore involved in various diseases, ranging from skin blistering to cancer metastasis.

Although the understanding of keratin network establishment, maintenance, modification and regulation is a key factor in elucidating the molecular mechanisms of keratin function in epithelial physiology, the details of keratin plasticity and organization in cells are largely unknown. This is because the resolution of light microscopy is not sufficient to resolve many of the details of IF. With the advent of super-resolution microscopy we now have at least 10-fold improved resolution. Here we will use the ground state depletion (GSD) technique (Fig 1) to unravel structural details of the keratin intermediate filament system that are not accessible by traditional light microscopy techniques. We will follow up on several very promising observations that we have made in preliminary experiments and hope to publish these findings within a year.

The Netherlands Cancer Institute / Anthoni van Leeuwenhoekhuis (NKI/AVL) is a world-class comprehensive cancer hospital and research institute. Within the NKI, in a dozen different departments many different aspects of basic cell biology, genetics, oncogenesis and cancer treatment are studied. In the department of Cell Biology we compare tumor cells to their untransformed counterparts, with emphasis on (regulation of) cell division and locomotion. Our group is specialized in applying advanced live-cell imaging techniques, FRET, FLIM and super-resolution imaging to these research questions. The current project is under guidance of Leila Nahidi MSc and prof. dr. Kees Jalink, in the department of cell biology.

Technical skills/methods: Cell culture, transfections, live-cell confocal microscopy,stimulation with agonists, SR microscopy, TIRF microscopy, image analysis, make hi-end preparations. You will participate in all group meetings and attend weekly world-class talks in the NKI lecture series.

Timespans: >36 EC = 24 weeks

Remarks: Because of the speed of developments, it is inpossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.

Contact: Kees Jalink,  tel.: 31-20-512-1933/1947 NKI-AVL Plesmanlaan 121 Amsterdam

 

Building novel multi-colour lineage tracing reporters
Research organization:  LCAM-FNWI, University of Amsterdam
Institute: SILS
Department: section of Molecular Cytology
Examinator: dr. Renée van Amerongen
Supervisor: Anoeska van de Moosdijk , dr. Renée van Amerongen

Description:

Despite the continued improvement of in vitro model systems, in vivo models remain required to fully capture the dynamic complexity of mammalian development and tissue homeostasis. Genetically engineered mouse models are indispensable i) for dissecting how tissues are built and maintained and ii) for unraveling the mechanisms behind cancer initiation and progression. For instance, inducible lineage tracing studies have revealed the complexity and plasticity of the mammary epithelial stem cell compartment.

Most inducible mouse models for lineage tracing use CreERT2/loxP technology. Here, spatiotemporal control over gene (in)activation is provided by combining a tissue-specific promoter with the administration of tamoxifen. Because it was found that high levels of tamoxifen can influence mammary gland growth, we want to switch to an rtTA/tetO-Cre/loxP system.

Lineage tracing has recently become a new golden standard for following cell proliferation through complex structures such as 3D cultures or whole tissue preparations. However, the current multi-colour models have limitations that make them unsuitable for some experiments. We want to build a novel multicolour tool to monitor stem cells in Wnt-responsive tissues such as the mouse mammary gland.

Since we are always looking for ways to build better tools and mouse models to monitor stem cells and Wnt signalling, you will test several (multi)colour constructs in vivo in cell lines to determine what multicolour construct to use for embryonic stem cell targeting. Depending on efficiency, targeting will be done via traditional gene targeting or CRISPR/Cas9 technology.

Depending on your interests, you will

  • Generate various fluorescent proteins with either membrane- or nuclear localization signals and test these for use in vivo.
  • Clone and test functionality of a multicolour reporter construct in mouse embryonic stem cells.
  • Perform targeting in mouse embryonic stem cells and screen for successful targeting events.
  • Compare Cre/Lox switching efficiency and leakiness for 2 distinct systems: rtTA/tetO/Dox and CreERT/Tam.

 

 Technical skills / methods: CRISPR technology, gene targeting, DNA cloning, (embryonic stem) cell culture, confocal microscopy, Western Blot analysis

Timespans: >36 EC = >24 weeks

Contact: Renée van Amerongen, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Optimizing fluorescent probes to develop a next generation highly sensitive FRET based biosensors
Research organisation: LCAM-FNWI, University of Amsterdam
Institute: SILS
Department: section of Molecular Cytology
Examinator: dr. ir. Joachim Goedhart
Supervisor: Marieke Mastop

Description: 

In our lab G-protein coupled receptor (GPCR) signaling is studied using FRET based biosensors. The aim herein is to elucidate what determines the specificity in GPCR signaling. In order to gather quantitative kinetic data on signaling profiles, highly sensitive FRET based biosensors are required. The quality of a FRET pair in such a biosensor is amongst others affected by:

  1. Spectral overlap of donor emission and acceptor absorption
  2. Molar extinction coefficient of the acceptor
  3. Distance between the fluorescent proteins (FPs)
  4. The relative orientation of the FPs
  5. Quantum yield of the donor
  6. Photostability of FPs

To alter the relative orientation of two FPs, one can apply circular permutation. This implies the joining of the N- and C- termini and introduction of new termini at other sites in the β barrel. In our lab a circular permutated variant of yellow fluorescent protein, cpV6, is often used as FRET acceptor. This circular permuted variant results in more efficient FRET in the “high FRET state” of the sensor, which increases the contrast of the FRET sensor.

Technical skills/methods:

During this project you will construct circular permutated variants of frequently used GFP variants. Promising variants will be applied in G protein activation biosensors. This project involves molecular biology techniques like cloning and cell culture as well as advanced imaging techniques as FRET, FLIM and FCS.

Timespan: >30 EC = >20 weeks 

Contact: Marieke Mastop, Molecular Cytology, University of Amsterdam Sciencepark 904, 1098 XH Amsterdam, The Netherlands

 

Development of live-cell super-resolution microscopy to unveil LPA receptor clustering and endocytosis mechanism
Research organisation: LCAM-NKI, Netherlands Cancer Institute
Department: Cell Biology
Examinator: dr. Kees Jalink
Supervisor: dr.  Kees Jalink & Daniela Leyton Puig

Description:

Stochastic localization super-resolution microscopy techniques for live-cell imaging require the use of special photo switchable fluorophores that are usually switched-on with the use of 405nm lasers. The photo activation and the excitation of fluorophores for super-resolution require high irradiation intensities to induce the characteristic necessary blinking of these fluorophores. Such high photon densities are likely to induce cellular damage in live-cell experiments. In fact, a recent study determined that while cells stand light intensities of ~1 kW cm−2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm−2, emphasizing the need for red fluorophores for live-cell localization microscopy. The group of prof. dr. Dorus Gadella has been developing improved red fluorescent proteins that don’t need activation or switching laser and work for super-resolution microscopy.

The heptahelical G protein-coupled receptor (GPCR) family includes more than 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane. GPCR internalization functions to control signal termination and propagation as well as receptor re-sensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo.

Technical skills/methods:

We will use the newly developed RFPs with some blinking proteins in other colors to study the regulation of LPA receptor 1 after stimulation with its ligand and other compounds, using live cell super resolution microscopy.

Timespans: >36 EC = 24 weeks

Remarks: Because of the speed of developments, it is inpossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.

Contact: Kees Jalink,  tel.: 31-20-512-1933/1947 NKI-AVL Plesmanlaan 121 Amsterdam

 

Using super-resolution microscopy to unveil LPA receptor clustering and endocytosis mechanism
Research organisation: LCAM-NKI, Netherlands Cancer Institute
Department: Cell Biology
Examinator: dr. Kees Jalink
Supervisor: dr.  Kees Jalink & Daniela Leyton Puig

Description:

The heptahelical G protein-coupled receptor (GPCR) family includes more than 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane. GPCR internalization functions to control signal termination and propagation as well as receptor re-sensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo.

We will use super-resolution microscopy to study the regulation of LPA receptor 1 (LPAR1) after stimulation with its ligand and other compounds.

The Netherlands Cancer Institute / Antoni van Leeuwenhoek ziekenhuis (NKI/AVL) is a world-class comprehensive cancer hospital and research institute. Within the NKI, in a dozen different departments many different aspects of basic cell biology, genetics, oncogenesis and cancer treatment are studied. In the department of Cell Biology we compare tumor cells to their untransformed counterparts, with emphasis on (regulation of) cell division and locomotion. Our group is specialized in applying advanced live-cell imaging techniques, FRET, FLIM and super-resolution imaging to these research questions. The current project is under guidance of Daniela Leyton Puig MSc and prof. dr. Kees Jalink.

 

Technical skills/methods:  Cell culture, transfections, cloning, live-cell confocal microscopy, SR microscopy, TIRF microscopy, image analysis. You will participate in all group meetings and attend weekly world-class talks in the NKI lecture series.

Timespans: >36 EC = 24 weeks

Remarks: Because of the speed of developments, it is inpossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.

Contact: Kees Jalink,  tel.: 31-20-512-1933/1947 NKI-AVL Plesmanlaan 121 Amsterdam

 

From meters to nanometers: Using super resolution imaging to visualize condensed DNA
Research organisation: LCAM-NKI, Netherlands Cancer Institute
Department: Cell Biology
Examinator: prof. dr. Kees Jalink
Supervisor: prof. dr.  Kees Jalink & Dr. Ahmed Elbatsh

Description:

When cells divide, it is crucial that the two newly formed daughter cells harbour the exact same genetic material as their mother cell. It is quite a challenge for dividing cells to separate the several-meters long DNA threads without any errors. To achieve this, cells condense their long threads of DNA into short and compact structures that are only few microns in length. This condensation is essential for faithful segregation of DNA but the responsible mechanism(s) are still a big mystery.

 

Condensin is an ATPase enzyme that is known to drive the DNA condensation process. The condensin complex consists of five proteins, which together form a gigantic ring-shaped structure that can entrap DNA threads. One model of how condensin confers condensation is by forming and stabilizing long DNA loops inside its ring, thereby bringing distant parts of the chromosome closely together. Through the formation of many consecutive loops, DNA threads would thus become condensed. In this project we aim to visualize DNA and condensin by optical super resolution (SR) microscopy https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2014/press.html

 

In SR microscopy the diffraction limit which hampers conventional fluorescence microscopy is circumvented and thereby a resolution up to ~10 nm can be achieved. The first goal is to optimize SR microscopy of DNA and condensin in fixed cells. We will then study how condensed DNA is shaped and the localization of condensin on condensed DNA. We will make use of a number of different condensin variants that either impair or enhance the condensation process. Comparing those different variants of condensin by super resolution might provide valuable insights into how DNA condenses.

 

Technical skills/methods:  In this project, the groups of Dr B. Rowland and Prof. Dr. K. Jalink, both at the division of Cell Biology, team up. Techniques used include: cell culture / transfections / confocal microscopy / SR microscopy / computer image analysis

Timespans: >36 EC = 24 weeks

Remarks: Because of the speed of developments, it is inpossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.

Contact: Kees Jalink,  tel.: 31-20-512-1933, k.jalink@nki.nl or Dr. Ahmed Elbatsh, tel: 020 512 2095, a.elbatsh@nki.nl

 

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