Single-cell Physiology

Below, you can find the updated list of Bachelor and Master internships available at the moment.

For more information on available internships in the Single-cell Physiology Team, please contact directly the teamleaders:

Evelina Tutucci – evelina.tutucci@vu.nl

Johan van Heerden – j.van.heerden@vu.nl

Project titleType of researchSupervisor(s)
Fishing for mRNA spatial patterns in Fission YeastExperimental (Master)Evelina Tutucci (evelina.tutucci[at]vu.nl)
and Johan van Heerden (j.van.heerden[at]vu.nl)
Background
Gene expression is a highly regulated process in all organisms, from bacteria to man. While we know many of the details of gene expression regulation, a largely unexplored phenomenon is the role that mRNA localization plays. There are indications that the regulation of mRNA localization can function as an additional layer to control protein activity. For example, many proteins function in specific sub-cellular locations, and translating such proteins in close proximity to their final destination would promote their activity. In contrast, translation at distant cellular locations would serve to delay activity. In this view, spatial regulation of mRNA provides a means to temporally control the activity of proteins.

In eukaryotic cells, the cell cycle is a complex process involving many proteins that are expressed in a specific order. Here the timing of expression of each protein is particularly important to ensure progression from one cell cycle stage to next. Whether mRNA localization could function an as additional regulatory layer to control the timing of cell cycle progression, is an open question. As a first step towards answering this question, in this project we will investigate whether the transcripts of genes involved in cell cycle regulation exhibits spatial organization in the fission yeast, Schizosaccharomyces pombe.

Approach
In this project you will use single-molecule FISH (smFISH) to study and quantify the spatial distribution of mRNA's of genes involved in cell cycle-regulation, during the cell cycle of fission yeast.

You will apply basic micro- and molecular biological techniques, in addition to more advanced microscopy and image-processing procedures.

Outcome
mRNA localization patterns will be used to identify candidate genes for further investigation into the role of spatial organization in gene expression regulation.

Your CV
As this project will involve a lot of computation and image and data processing, ideally you have some affinity with programming (R, Python, Java or Matlab).

Number of positions available
1

Duration of project:
5 months

Start date
~Oct 2022


Developing smRNA FISH for the identification of antibiotics-tolerant persister bacteriaExperimental (Master)Evelina Tutucci (evelina.tutucci[at]vu.nl)
Wilber Bitter (w.bitter@vu.nl)
Frank Bruggeman (f.j.bruggeman@vu.nl)
Motivation
Multi-drug-resistant, pathogenic bacteria threaten human health. They have acquired DNA mutations that make them insensitive to antibiotics. Pathogenic bacteria are also successful in dealing with antibiotics, and the human immune system, because they often can switch to a non-growing, antibiotics-tolerance state, called the ‘persister’ state. Persister cells are formed from growing bacteria, can switch back to the growing state and coexist with growing cells in cell cultures. Since persister cells are not growing, they can withstand the effects of antibiotics that kill growing cells. It has been shown that growing cells can switch into persister cells, via so-called toxin-antitoxin systems. Such systems have been discovered in Escherichia coli and have since been found in many pathogenic bacteria, including Pseudomonas aerogenosa and Mycobacterium tuberculosis. Understanding how persister cells form is an important research topic.
Aim of this project
Since persister cells derive from growing cells and coexist with them in growing cell culture, we require single-cell methods to identify persister cells. One promising method is to determine the relative number of toxin and antitoxin transcripts in single cells, as it is believed that an excess of toxins over antitoxins turn growing cells into persister cells. The aim of this project is develop single-molecule RNA FISH, which allows for counting of the number of transcripts in single cells, for identification of persister cells. First in Escherichia coli and subsequently in Mycobacterium segmatus, a relative of M. tuberculosis.
Methods and techniques
fluorescence microscopy, molecular biology, bacterial cell cultivation, RNA methods, smRNA FISH.
Impact
During this project you become familiar with state-of-the-art methods of microbiology, molecular biology and fluorescence microscopy that are used throughout molecular biology labs in hospitals, academia and industry. Since smRNA FISH is not routinely used in such labs, the expertise which you acquire during this project will count on the job market, regardless whether this concerns a fundamental or applied job.
Number of positions available
1
Duration of project
6 months
Starting date
As soon as possible
FILLED until October 2022
Visualizing and manipulating single-cell signalling in budding yeastExperimental (Bachelor or Master)Dennis Botman
d.botman@vu.nl
Yeast continuously adapts to changing conditions. To measure environmental changes and adapt adequately, yeast cells use signaling cascades, such as the cAMP-PKA pathway. In this internship, you will use our existing cAMP and PKA biosensors, optimize optogenetic tools to manipulate cAMP levels in cells and try to develop novel sensors to study how the cAMP-PKA cascade affects yeast physiology at a single cell level. Lastly, you will try to setup a CRISPR/CAS protocol to make genetic changes (in the cAMP-PKA signalling pathway) in our yeast strains.
Filamentous Growth and Biofilm formation in S. cerevisiae and C. albicans resolved by single cell imaging Experimental (Bachelor or Master)Evelina Tutucci (evelina.tutucci@vu.nl)
Background
Many fungi such as Saccharomyces cerevisiae or Candida albicans are able to switch between a unicellular (yeast) form to a multicellular filamentous form in response to changes in the environment (e.g. nutrients availability, stress). This morphological transition allows fungi to adopt different survival strategies and in some instances become pathogenic.
During filamentous growth cells acquire an elongated shape and unipolar budding pattern, allowing for greater exploration of the environment. A more advanced strategy is the formation of biofilms, multicellular structures that consist of different cell types (both yeast form and filamentous form) as well as an extracellular matrix, offering both increased structural integrity and resistance to antifungal drugs. While many of the genes required for this differentiation process have been identified through bulk analysis (e.g. RNA seq), their expression in single cells and during differentiation has remained largely unstudied.
Aim
In this project, we investigate at the single cell level, the gene expression changes occurring during fungal differentiation. By using a fluorescence-based RNA imaging technique called smFISH (see pictures: https://www.tutuccilab.com/) we visualize and quantify individual mRNA molecules in single cells to investigate the spatiotemporal control of gene expression during filamentation. Furthermore, we investigate how the spatial organization of cells in biofilms influences gene expression.
Planned activities (and methods)
During this project, you will learn how to cultivate filamentous fungi, various molecular biology techniques as well as smFISH and cutting edge fluorescence microscopy approaches. You will also be trained in imaging analysis, which will be used to investigate gene expression (RNA spot counts) of cells in biofilm. We will measure cell-to-cell heterogeneity and couple it with spatial information, cell volume and cell length in filamentous cells. Since the microscopy data is very rich of information it will be possible to further expand the analysis, depending on your computational skills and your curiosity.
A prior familiarity with programming languages such as Python and R is a must . You will participate and present in our Single-cell group meeting and Journal club.
Duration
6 Months
Starting date
From July 2022
From July 2022
Characterizing the newest fluorescent proteins in budding yeastExperimental (Bachelor)Dennis Botman
d.botman@vu.nl
Fluorescent proteins (FPs) are excellent tools to study organisms at a single-cell level. In order to obtain to best out of the available FPs, we would like to test the newest FPs in yeast. In this internship, you will test various new FPs on characteristics such as brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Among the new FPs, we hope that we find better variants that can improve our experimental signal readout, opening up new experimental possibilities.