Project topics and materials
Get Complete Project Material File(s) Now! »
RhoGTPases during cell migration
The Rho family
The Rho small GTPases family (RhoGTPases) (Table 1.1) is constitued by 21 mem-bers (Boureux et al. 2007) in humans of which importance for cell migration may vary depending on the cell type (Ridley 2015). But we can highlight the major importance of three proteins of this family, RhoA, Rac1 and Cdc42. They are described as being key-players for the actin remodeling at the front edge of a cell protrusion but also to play a role for the polarization of the cell that will allow directionnality in the cell migration. They are also among the most well studied protein of the family as they are the better conserved between species.
They all promote actin remodeling but with different modalities. Rac1 and Cdc42 are actively promoting actin branching via ARP2/3 complex whereas RhoA favor actin nu-cleation and elongation via mDia but also acto-myosin contraction. The general spatial description of each GTPase is to associate Rac1 and Cdc42 with the front of the migrat-ing cell whereas RhoA is linked to rear of the cell (Figure 1.5).
General localisation of the RhoA at the rear and Cdc42, Rac1 at the front. Even though reality is more subtle this give a general picture of the assymetric distribution of Rho GTPases. (From Mathieu Coppey Unpublished)
But reality is more complex, RhoA is also present in the protrusion but at a different location than Rac1 and Cdc42. Whereas RhoA is located at the tip of the protrusion, Cdc42 and Rac1 are located just a few m behind (Machacek et al. 2009). Their regula-tion is complex as they can be activated by different activators and themselves actuates on different targets. Rac1 and Cdc42 are also crossregulating eachother (Burridge et al. 2004; Beco et al. 2018; Nobes et al. 1999).
The regulation of the activity of the small GTPases is similar inside the family. It rely on a conformational change to an active form upon binding to GTP and the conversion of GTP to GDP to go back to its inactive form. Both events are catalysed by external protein as they are relatively slow otherwise (Boureux et al. 2007). Guanine exchange factors (GEF) catalyse the exchange of the GDP by GTP and are thus considered as ac-tivator of the Rho small GTPases. Guanine activating protein (GAP) is the inhibitor and favor the hydrolysis of the GTP to GDP to go back to the inactive form (Figure 1.6).
Regulation of the activity of the Rho family small GTPases. GEFs are catalyzing the exchange of GDP to GTP whereas GAPs catalyse the hydrolisis of the GTP to GDP. Under its GTP bound form the GTPase can bind effector proteins.
Many GEFs and GAPs have been described for each RhoGTPase some beeing more or less specific. For example, Rac1 can be activated by Tiam1, -PIX, and Dock180 (Ridley 2015). But -PIX has also been shown to be a GEF for Cdc42 and only Tiam1 is rather specific of Rac1. On its side Intersectin1 is considered to be more specific of Cdc42.
Once activated, RhoGTPases are able to form complexes of proteins that among the dif-ferent effect can recruit the ARP2/3 complex which allows actin branching. This allows the nucleation of new actin filament that can push the membrane forward. Cdc42 acts through the WASP complex and Rac1 interacts with the WAVE complex. RhoA on its side is more responsible of retraction linked to recruitment of the ROCK protein which activates Myosin motors, but also play a role in actin nucleation.
Our work has focused on Cdc42 and Rac1 activity using GEFs mostly specific for each of them. But we also tested more upstream activity regulators like PI3K.
Intersectin: A GEF for Cdc42
Intersectin1 (ITSN1) (O’Bryan 2010) is a multi target protein. It regulates Cdc42 through it’s Dbs homology (DH) domain(Hussain et al. 2001). The other domains have Itsn1 interactions with other proteins. If we consider only actin cytoskeleton remodeling activity, it has been shown to interact with Cdc42 through the DH domain, N-WASP and SOS through the SH3 domain and WIP through the EH domain (From interactions described in Herrero-Garcia et al. 2017)
Itsn1 domains scematic. The SH3 domains play a role in the autoinhibition of the GEF. The PH domain is necessary for the membrane localization. ( Inspired by Herrero-Garcia et al. 2017 )
been shown to allow interaction with other proteins but also to regulate the activity of the DH domain. The absence of the Pleckstrin homology (PH) domain is reducing the catalyzing activity because of its potential action on the conformation of the DH domain but also because the PH domain is expected to favor the membrane localization of the GEF by interacting with the lipids of the membrane (Whitehead et al. 1999; Hussain et al. 2001). Moreover, intersectin could inhibit itself through its SH3 (Zamanian et al. 2003) domain by interacting with the DH domain. This was proved by the fact that morphological changes appearing through the over expression of DH domain of ITSN1 Tiam1 exhibits a DH-PH domain for the regulation of Rac1 but also different regulatory domains as PHn-CC-Ex domain wich seems important both for regulation and localiza-tion.
that can be reversed by the expression of the SH3 domain (Kintscher et al. 2010). To lift this autoinhibition, it is suggested the necessary participation of other proteins of which Numb could be a candidate (Nishimura et al. 2006).
Tiam1: A GEF for Rac1
Tiam1 is as ITSN1 a GEF but specific of Rac1. It’s a multidomain protein that is describe to be mostly specific to Rac1 but that can also regulate other proteins through other interactions. It carries two PH domains one in C-terminal downstrean of DH domain which serves the same purpose as the one of ITSN1 and a PH domain in N-terminal which seems to auto inhibit Tiam1 (Mertens et al. 2003). This inhibition could be lifted by interaction with different proteins like CD44, Par3 or ephrin. (Xu et al. 2017)
But the PHn domain together with the CC-Ex subpart has also a positive role for the localization of Tiam1 (Stam et al. 1997). It presents a high affinity for phophorylated inositides which favor the localization at the membrane to activate Rac1.
GEF regulation
There exists many more GEFs. They are divided in two families, the DH-PH family and the DOCK family. Their regulation is based on many post translational modification as phosphorylation, acetylation, ubiquitynilation. The member of the DH-PH subfamily are also regulated by phosphoinositisides lipids (PIP) as PIP serve the GEFs to relocate at the membrane.(Mertens et al. 2003)(Hodge et al. 2016)
This last regulation is linked to a higher affinity of PH domains for more or less phos-phorylated PIP. A process which is regulated by PI3K. Making this last one an upstream regulator of the GEFs.
PI3K: Phosphoinositol regulation of GEF activity
As said earlier the PH domain of GEFs has an affinity for different kind of lipids. A subset of GEFs has a higher affinity for Phospho inositol(PI) 3,4,5 Phosphate (PIP3) and/or PI 4,5 Phosphate (PIP2). The phosphorylation of PI are regulated among other enzyme by the Phopho inositol 3 kinase (PI3K) of class 1 (Fruman et al. 2017). It acts through its Ish2 domain. The effect will vary among the different GEF, for some they seem to be just an anchor point like ITSN1, for others they increase the GEF activity like for Tiam1 and P-rex (Campa et al. 2015).
Intracellular pathways
From this descriptive point of view about molecular signals, we highlight that there is many different step of signal transmission which are modulated by many regulatory step. For decades attempts have been made for understanding the mechanisms of regulation of this cascade of proteins. The work is still ongoing and an effort is now done to understand how the information is processed through these pathways to output the correct choice for the cell.
These efforts to get a better understanding of the intracellular pathways were done using various perturbation techniques which are discussed in the following section.
Perturbation of intracellular pathways
The exploration of the intracellular mechanisms relies on many techniques. They all offer benefits and have allowed to explore different aspect of biological systems. Some have the capability of probing a cell population whereas other are suitable of single cell experiments. Here we review different approches that brought valuable information on intracellular pathways and we will conclude presenting our approach for this project.
Manipulation of cell functions: Multicellular approach;
Functional genomics
Around the 80’s, the discovery of restriction enzymes and the development of Poly Chain reaction (PCR) mark the start of gene editing. Deletion, point mutation, gene fusion permitted gain or loss of function in protein and opened the path to study intra-cellular mechanism at the protein level. Not only, these techniques allowed to highlight the role of a protein in a specific function but also the role of its subdomain in executing and regulating its functions. The continuous improvement of gene editing tools but also of high throughput techniques in gene sequencing produced a large part of the description of protein family through the constitution of large proteomic databases that allow us to relate proteins in between them and to have an overview of their main functions.
Concerning RhoGTPases, the role of gene editing tools were valuable. It allowed to link together the different small Rho GTPases in families sharing functions (Figure 1.1) through gene conservation studies. It also pointed out the similarities between their acti-vators, the GEFs, and their inhibitors, the GAPs.
Through mutations of the GEF, the conserved role of their domains have been shown. For example, the common role of the DH-PH for the interaction with the GTPases. It also highlighted the role of specific amino acids for the specificity of interaction both for their effector targets as well as for their lipids affinity through their PH domain. It also revealed the importance of the other subdomains of the proteins for their regulation or alternative mode of action (Mertens et al. 2003; Stam et al. 1997)
Finally, mutations have allowed to understand the role of this proteins through consti-tutively active (Q61L) form or inactive forms (T17N) for Rac1 (Revach et al. 2016) and Cdc42. The study of these mutants also helped to understand anomalous behavior of cancer cells that often upregulate these protein activity.
The development of gene editing opened the door to bioengineering of many sort of pro-tein that allow scientists to get control on intracellular protein activity. These different techniques to control intracellular proteins are discussed below. We will start discussing chemically induce dimerizer as they were the proof of concept that showed intracellular signal manipulations, then optogenetic and finally magnetically controlled signals using engineered proteins.
Drug induced dimerization
Different kind of drugs can be used to control cellular activity. One kind of drugs can interact with proteins to inhibit or enhance their activity, these are not discussed here. The kind we discuss is made of couples of chemical/proteins that have been used to create artificial functions in cells. Functions which are triggered by exposition to the corresponding chemical.
The main advantage of engineered systems is that in theory they have a low risk of in-teraction with preexisting signaling mechanisms of the cell. Such system appeared in the early 90s with the development of doxycycline dependent gene expression. First, with the tTa protein that promote gene expression in absence of doxycycline. Then with rTTA which induces gene expression in presence of the doxycycline molecule. Both were based on the fusion of the TetR protein from Erischerischia Coli with the activating domain V16 of the herpes simplex virus(Gossen et al. 1995; M. Gossen et al. 1992). It offered the ability to control gene expression in a reliable manner to study the role of genes in spe-cific mechanisms. The downside of this system is that it needs a delay before the desired gene is expressed. It also requires some time before the protein gets degraded. Together these issues limit the possibility to study the effect of fast on-off switch of a protein ac-tivity.
At about the same time, the discovery of the FRB fragment that bind the FKBP-rapamycine couple offered a tool to control the dimerization of proteins (J. Chen et al. 1995). In practice, two proteins can be engineered one carrying the FRB fragment and the other fused to FKBP.
These two proteins can then dimerize upon addition of rapamycine with a high affin-ity (Kd in the nM range). This solved the problem of having a fast activation but it result that the activation if not easily reversible. Still using this strategy Inoue et al. 2005 showed that it was possible to use the DH-PH domain of a GEF to manipulate Rho GTPase activity. Tiam1 DH-PH domains were fused to FKBP and a second protein was a fusion of FRB and Lyn11. The later is a peptide targeting the plasma membrane. On addition of Rapamycine analogues they could increase the formation of lamellipodia in the cell expressing the previous constructs. The coexpression of a reporter PAK1-YFP confirmed that this effect was mediated through an increase of activity of Rac1.
Single cell manipulations
Even if the aforementioned techniques were efficient to explore intracellular mecha-nisms they suffer from from the possibility to generate fast (in minute time scale) on-off stimulation of intracellular signaling proteins. Genetic modification produce a permanent effect once it is applied, thus no live interaction can be produced. Chemically induced mechanisms allow to turn on an engineered mechanisms but the reversal of these events can take as long as the time necessary to degrade the affected proteins.
To study the dynamics of intracellular event, it is required to have access to a tool that can be modulated spatially and temporally in minute timescales. Here, we start to present first the optogenetic tools that represent the gold standard for intracellular protein manipulations. On the following, we will then move to the strategies implying magnetic manipulation and explain the chosen strategy for this work.
Light based signaling manipulation
There is a wide range of tools based on light that allow to experiment on single cell either to test their physical parameters, like optical tweezers or to modulate protein ac-tivity. We focus here on the manipulation of proteins using photosensitive proteins. This category of tools are grouped in a family named optogenetics.
All of them are based on light sensitive proteins discovered in different species that have been successfully used to manipulate intracellular mechanisms using light. Two groups can be defined. The first is based on opsin proteins which are sensitive to light. The channel rhodopsin protein family identified in some bacterias and plants is a ionic chan-nel that allows the transfer of cations or anions (depending on the channel type) through the membrane (Nagel et al. 2003). This is a useful tool to study all phenomenon based on electric polarization of the plasma membrane of the cell. Other opsin proteins regulating G protein (also know as guanine binding proteins) activity, have also been identified in many species. Endogenously these last are responsible for light detection in mammalian (Karunarathne et al. 2015).
The second group allows the manipulation of protein thanks to couple of dimerizing pro-teins that have been identified over the last decades. Starting with PIF3/PhyB (Ni et al. 1999; Shimizu-Sato et al. 2002) discovered in Arabidopsis is photoactivated with red light and disactivated with far-red light. As precise control of the dimerization requires the use of two wavelengths making it complicated to use. Moreover, PhyB also requires a chromophore which is not available endogenously in mammalian cells. Blue light sensi-tive proteins are represented by the CRY2/CICBN couple (Kennedy et al. 2010) and the iLID/SspB couple (Guntas et al. 2015). All these proteins offer a wide panel of tools with different properties. For example just variants of iLID/SspB offer different affinity, from nM to M range in the dark to hundred of nM and M range respectively in the light (Zimmerman et al. 2016).
Also, for these two couples of proteins, the dissociation of the dimers once put back in the dark is in the range of minutes (Spiltoir et al. 2019). Their binding half life range from hundred of seconds for iLID/SspB to 5 minutes for CRY2/CIBN.
Table of contents :
1 Introduction
1.1 Generalities
1.1.1 Magneuron project
1.1.2 RhoGTPases during cell migration
1.1.2.1 The Rho family
1.1.2.2 Intersectin: A GEF for Cdc42
1.1.2.3 Tiam1: A GEF for Rac1
1.1.2.4 GEF regulation
1.1.2.5 PI3K: Phosphoinositol regulation of GEF activity
1.1.3 Intracellular pathways
1.2 Perturbation of intracellular pathways
1.2.1 Manipulation of cell functions: Multicellular approach;
1.2.1.1 Functional genomics
1.2.1.2 Drug induced dimerization
1.2.2 Single cell manipulations
1.2.2.1 Light based signaling manipulation
1.2.2.2 Magnetic control
1.3 Developping manipulation of intracellular magnetic objects
1.3.0.1 Diffusion of particles in the cytoplasm
1.3.0.2 Limitation to diffusion in the cytoplasm
1.3.0.3 Measuring diffusion in intracellular space
1.3.1 Particles stability
1.3.1.1 Colloidal stability
1.3.1.2 Passivation
1.3.2 Magnetic forces on nanoscale magnetic objects
1.3.2.1 Magnetic particles characteristic and Magnetic field
1.3.2.2 Manipulation of proteins inside the cytoplasm
1.3.3 Specific targeting of proteins
1.3.4 Final words
2 Material and methods
2.1 Cellular biology
2.1.1 Cell lines
2.1.1.1 Hela CCL2 (Hela)
2.1.1.2 Retinoid Pigmental Epithelium hTERT (RPE1)
2.1.1.3 human Mesenchymal Stem Cells (hMSC)
2.1.1.4 SH-SY5Y cells
2.1.2 Medium
2.1.2.1 DMEM medium
2.1.2.2 DMEM/F12 (1:1) medium
2.1.3 Transient transfection
2.1.4 Stable cell lines
2.1.5 Coverslip coating
2.2 Micropatterning
2.2.1 Coverslip cleaning
2.2.2 Passivating molecule: PLL-g-PEG
2.2.3 Passivating molecule: PAcrAm-g-(PMOXA,NH2,Si)
2.2.4 Micropatterning: Quartz mask approach
2.2.5 Micropatterning: photopattering approach
2.2.6 Incubation with protein
2.2.7 Cell platting
2.3 Molecular biology
2.3.1 Gene cloning
2.3.2 Plasmid list
2.4 Microscopy
2.4.1 Olympus inverted microscopes
2.4.2 Nikon inverted microscope
2.4.3 Optogenetics: DMD equiped microscope
2.4.4 Heating system
2.5 Particles
2.5.1 Silica particles
2.5.2 Ferritin
2.5.2.1 Ferritin: Protein purification
2.5.2.2 Protein expression
2.5.2.3 Purification : protocol
2.5.2.4 Purification : Histag
2.5.2.5 Core synthesis
2.5.3 Quantum Dots
2.5.4 Other particles
2.6 Magnetic configuration
2.6.1 Iron tips
2.6.2 Micro array
2.7 Internalization of particles
2.7.1 Microinjection
2.7.2 Electroporation
2.7.3 Pinocytic loading
2.8 Micromanipulation: attraction of the particles
2.8.1 Magnetic tips
2.8.2 Micromagnets
2.9 Analysis and quantifications
2.9.1 Intensity measurements
2.9.2 Protrusion growth
2.9.3 Single Particle tracking
2.9.4 SPT: analysis
2.9.5 Cell mapping
3 Results
3.1 Exploration of cytoplasm environment and particles diffusion properties
3.1.1 Behaviors of particles below pore size
3.1.2 Zwitterionic Quantum dots
3.1.2.1 Diffusion of zwitterionic coated particles in the cytoplasm
3.1.2.2 Efficient targeting using Biotin-Streptavidin strategy
3.1.3 Exploration of intracellular cytoplasm
3.1.3.1 Diffusion in full cells
3.1.3.2 Exploring intracellular space: what’s next?
3.2 Stability of magnetic particles
3.2.1 Behaviors of magnetic particles
3.2.1.1 Si-MNPs
3.2.1.2 fMNPs
3.2.2 Parallelization
3.2.2.1 Note on the development of parallelized manipulation
3.3 Magnetic manipulation of intracellular signals
3.3.1 Manipulation of ITSN1 DH-PH domain
3.3.1.1 State of the technic: a successful manipulation
3.3.1.2 About the dynamics of the observed events
3.4 Exploring the limits of magnetic intracellular manipulation
3.4.1 Low affinity
3.4.2 Upstream signal activation: Ish2
3.4.3 Crowding
3.4.4 Discussion on magnetic manipulation
4 Conclusions and perspectives
4.1 Manipulation of intracellular mechanisms with the magneto-molecular approach
4.1.1 Magnetic particles
4.1.2 Magnetic manipulation
4.1.3 Signaling proteins
4.1.4 A clearer view on the cytoplasm
4.2 Final words: Controlling cells mechanisms
