Quantitative single molecule imaging in living cells and animals

Speaker: Christof Gebhardt

Department: Institute of Biophysics, Universität Ulm

Subject: Quantitative single molecule imaging in living cells and animals

Location: Bionanoscience Department, TU Delft

Date: 07-04-2017

Author: Mirte Golverdingen

Life is an interplay of inanimate biomolecules, Christof Gebhardt tries to understand how these biomolecules function in isolation, interaction and what happens with them when we are ill. To understand this the research to reaction kinetics, their spatial distribution, stoichiometry and forces can help. Moreover, biomolecules can be better understood if we manipulate single biomolecules in vitro and in vivo.

Super resolution methods in combination with fluorescent proteins as markers can help us to understand biomolecules better. Gebhardt uses super resolution in combination with GFP. Next to green colours, other colours of the fluorescent protein are developed. However, single GFP expression for single molecule detection is hard to visualize using fluorescent microscopy because of the fluorescent background.

HILO illumination is an imaging method that only measures a small part of the cell, in this way the background can be reduced.  By adding a mirror to the HILO set-up, the side of the object can be imaged and in this way, you have more space to alter the cell at the top (See Figure 1). Moreover, imaging from the side reduces the contrast. The 3D image of the cell can be imaged by moving the objective up and down. By using other tags than GFP, Gebhardt was able to use HILO for in vitro, in cellula, in vivo and in silico studies.

Honours9

Figure 1: Scheme of the reflected light-sheet principle (not drawn to scale). A laser beam is focused by an objective to form a vertical light sheet that is reflected by 90° off an AFM cantilever next to a cell in a Petri dish. Fluorescence is detected by a second high-NA objective. Three-dimensional optical sectioning is achieved through vertical displacement of the sample. Adapted from: Gebhardt, J. C. M., et. al (2013). Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nature methods, 10(5), 421-426.

Gebhardt for instance, studied the transcription factor and DNA (TF-DNA) interaction times in chromatin organisation and transcription regulation. The transcriptional repressor CTCF interacts with the chromatin, however, it is not known how CtCF interacts with the chromatin during the cell cycle. Moreover, the influence of the regulator role of TF-DNA interaction time on transcription repression is a research topic of the research group of Gebhardt.

Kinetic information from the data can be obtained by researching video images of with an HoloAR labelled nuclear receptors the diffusion coefficient, the DNA bound tag and the DNA residence time of molecules can be obtained. To overcome the problem of photobleaching that occurs after continuous illumination, dark times were used. By measuring the DNA residence time of different TFs, Gebhardt showed that every transcription factor has another DNA residence time. There can be made a differentiation between specific (1/10/100 s) and unspecific (<1) DNA binding residence times.

CtCF chromatin interaction kinetics

The 3D organization of chromatin exists of nucleosomes, looped gene loci and chromosome territories. The chromatin architecture influences gene expression, differentiation, development, DNA replication and DNA repair. However, the control of the DNA loops is still unclear.

Chromatin topology is mediated by architectural proteins. Gebhardt was able to calculate the topologically associating domain, this shows how likely it is in a loop if chromatins interact with each other. There are certain TFs that is able to interact with the architectural proteins. CtCF is one of these transcription factors, therefore, it is seen as a potential ‘master weaver’. However, it is not clear if CtCF is activating, repressing or insulating the architectural proteins. CtCF contains 11 zinc finger motives that are able to dimerize the DNA. It has more than 100000 target sites on the DNA and it recruites cohesion. Gebhardt his study focusses on the kinetics of the CtCF TF, how long does it bind to the chromatin? And is the TF stable or mobile?

He was able to design a method by using fluorescent proteins that shows a wide spread binding kinetics. By developing a method based on the measured fluorescence, Gebhardt showed that CtCF shows three dissociation states. The residence times of these states are: <1s, <10s and ~1000s. The last two are specific, while the first one is specific and occurring in G1 and G2 phase of the cell cycle.

CtCF has low- and high-affinity sites on the DNA and its dynamic interactions (~4s) are changing the loops within topological associating domains (TADs) or initiate abortive loop formation. CtCF chromatin interaction depends on the current phase of the cell-cycle. In the S-phase, much less high (stable) resistance times are measures, so in the S-phase an overexpression of CtCF blocks cells. It could be that replication dissolves CTCF-chromatin interactions during S-phase. In the M-phase: CtCF is excluded from chromatin, this may be caused by phosphorylation.

By studying the interaction of CtCF with the DNA, Gebhardt et al. showed that CtCF-chromatin interaction is cell-cycle dependent. Kinetic information on the interaction of biomolecules with DNA can help us understand the complex interactions in the cell better. I liked that he was able to adapt the traditional way of imaging with HILO to a more advanced and better adapted method. Moreover, the discovery of the tree dissociation states of CtCF with their corresponding residence times invites us to research the dissociation states of other transcription factors, giving more insight to the complex cell dynamics.

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Further investigation to the role of Arf1 in the Golgi apparatus

Speaker: Francesca Bottanelli
Department:
Yale University
Subject: Live-cell nanoscopy of protein sorting at the Golgi
Location: A1.100 (TU Delft)
Date: 14-11-2016

Kristian Blom

At the 14th of November I visited a BN seminar given by dr. Francesca Bottanelli, a postdoctoral associate in cell biology in the Rothman Lab at Yale University. Dr. Bottanelli is a cell biologist who is interested in the use of Stimulated Emission Depletion (STED) for living cell imaging. At the moment she uses STED to image COPI protein dynamics at the Golgi apparatus in mammalian cells. 

Dr. Bottanelli started the talk by introducing the main actor of her research: the Golgi apparatus. This organelle is the main sorting station of the cell and despite decades of research we are still far from fully understanding how it functions. Proteins that are synthesized in the endoplasmic reticulum are packaged into vesicles, which then fuse with the Golgi apparatus. Thereafter they are transported to the right location in the cell. While the machinery and molecular interactions involved in cargo sorting have been extensively investigated in vitro, there is a lack of understanding of the dynamics and nanoscale organization in living cells, since intracellular transport occurs over extremely short distances of a few 100 nm. A little progress is made over the past decade due to the difficulty to image molecular processes in the intrinsically crowded perinuclear area using standard diffraction limited imaging techniques.

After the introduction we went further into detail about STED. This imaging technique is part of the so called super-resolution microscope techniques, which means that the resolution that you can get with this technique bypasses the diffraction limit. In conventional fluorescence microscopy the electrons of a fluorophore are excited by incoming light of a certain wavelength, and thereafter by relaxation of the excited electron light of one specific wavelength is emitted back by the fluorophore (green arrow figure 1). However, with STED it is possible to force the excited electron into a higher relaxed vibration state than the fluorescence transition would enter, causing the released photon to be red-shifted (orange arrow figure 1). To make this alternative emission occur, an incident photon must strike the fluorophore. One can achieve super resolution by depleting fluorescence in specific regions of the sample while leaving a center focal spot active to emit fluorescence.

Figure 1 – A Jablonski diagram for the process of fluorescence and STED. Blue: Excitation of an fluorophore’s electron to a higher energy state. Green: Fluorescence – Relaxation of the excited electron. Due to the drop in energy light is emitted. Orange: STED – Relaxation of the excited electron to a higher energy state compared to the orange version. Due to the smaller energy drop during relaxation, light with a longer wavelength is emitted.

Dr. Bottanelli developed a novel labeling strategy for dual-color live-cell STED. Taking advantage of gene editing techniques (CRISPR/Cas9) and her novel live-cell STED labeling strategy she took a careful look at the role of ARF1, a protein localized in the Golgi apparatus that has a central role in the formation of COPI vesicles at the Golgi. The role of ARF is mainly to recruit adaptors and coat proteins for the formation of transport carriers. However, Dr. Botanelli found out that besides its role in generating COPI vesicles by recruiting Coatomer at the the Golgi, ARF1 also is involved in the formation of anterograde (from ER to Golgi) and retrograde (from Golgi to ER) tubular carriers.

Due to my lack of knowledge about the Golgi apparatus and the different proteins that are involved in the transportation process, it was rather hard for me to follow the talk. However, once I started to put everything on paper, more and more about what was told became clear for me. Afterwards I asked myself the question whether dr. Botanelli already found out if these anterograde and retrograde carriers are the main transportation systems that allow for the protein dynamics between the ER and Golgi apparatus. Since I did not followed everything during the talk, it could be that I missed that part. If that is not the case it might be a nice topic to do research about for in the future.