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Modulating L‐plastin function in immune cells by nanobodies : how small bodies can have large consequences

Sarah De Clercq (UGent)
(2013)
Author
Promoter
(UGent)
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Abstract
The actin cytoskeleton is a crucial part of both eukaryotic and prokaryotic cells. This can be taken literally, as it is the largest cellular system found in cells. Furthermore, the cytoskeleton is also crucial for the correct functioning of the cell, since it is in charge of the shape, polarity, motility, division, differentiation, contraction, adhesion,… of the cell. It goes without saying that, in order to fulfill this list of tasks, this structure has to be dynamic, always ready to adapt to the changing needs of the cell. However, as is often the case for rather cumbersome organelles, they rely on little, versatile players, to make them more dynamic. These are here represented by the so‐called “actin binding proteins” (ABPs), a plethora of actin polymerizing/depolymerizing, bundling/crosslinking, capping, monomer binding,… proteins which regulate the above mentioned processes. This thesis focuses (in descending order of importance) on the bundling protein L‐plastin (LPL), the severing and capping protein gelsolin (GSN), and to a lesser extent on the capping protein CapG. Lplastin (or leukocyte plastin) is exclusively expressed in hematopoietic cells and ectopically in several cancer cells. Gelsolin and CapG are both abundantly expressed in most cell types. Nanobodies (the variable part of camelid heavy chain only antibodies) targeting these three ABPs have previously been characterized in our lab and have proven to be bona fide antagonists of these proteins in cancer cells, counteracting their motility and invasion. In this thesis, these nanobodies were employed in another cell context, namely in immune‐related phenomena. In a first part of this thesis, Nbs targeting L‐plastin and gelsolin were used in a study concerning the formation of the immune synapse (IS) and subsequent T cell activation, an important event in the initiation of the immune response following insult by pathogens and toxins. L‐plastin has been shown before to play a role in the formation of the IS between antigen presenting cells (APCs) and T cells. A possible role for gelsolin has not been investigated before. The use of Nbs, targeting distinct domains of L‐plastin and gelsolin, makes it possible to reveal subtle mechanistic details as particular functions of these proteins can be silenced. One LPL Nb specifically interacts with the EF hands, thereby sequestering L‐plastin in an inactive conformation, preventing it from interacting with actin. Another LPL Nb binds to the actin binding domains of L‐plastin and interferes with actin bundling. When expressed in human T cells, both LPL Nbs perturbed IS formation as well as the polarization of the T cell toward the center of the APC, with subsequent docking of the microtubule organizing center (MTOC). Both Nbs also delayed L‐plastin phosphorylation, which is required for optimal bundling. One Nb reduced the interaction between L‐plastin and the integrin LFA‐1, which is important for the adhesion between T cells and APCs. Interaction of L‐plastin with LFA‐1 is thought to stabilize LFA‐1 at the immune synapse and indeed, LFA‐1 enrichment at the synapse was reduced in the presence of this Nb. Furthermore, this Nb delayed Ser745 phosphorylation of LFA‐1. Consequently, T cell activation was defective, as demonstrated by a hampered T cell proliferation and diminished interleukin‐2 (IL‐2) secretion. Although gelsolin was shown to be also present at the IS, a nanobody targeting the actin‐free population of gelsolin had no effect on immune synapse formation and subsequent T cell activation. In a second part of this thesis, L‐plastin, gelsolin and CapG Nbs were employed in a study concerning podosomes. These are dynamic adhesion and degradation structures, consisting of an actin‐rich core, surrounded by a ring of adhesion molecules. They are found in monocytic, smooth muscle and endothelial cells, as well as in transformed fibroblasts. L‐plastin and gelsolin have repeatedly been described to colocalize with F‐actin in the podosomal core, and are thought to fulfill important, yet distinct, roles in podosome assembly, turnover, stabilization and/or function. We further showed that, unlike the other 2 proteins, CapG is not enriched in podosomes of macrophage‐like THP‐1 cells. Also here, distinct nanobodies were used which revealed novel insights. A gelsolin nanobody specifically targeting active, actin‐bound gelsolin, was enriched in podosomes. This was not the case for a nanobody targeting actin‐free gelsolin. However, the presence of the above described L‐plastin nanobodies had a serious impact on podosome formation and function. Cells expressing these Nbs were not capable anymore of forming stable podosomes, demonstrating the (normal) regular and cyclic turnover of Factin was lost. Instead, this turnover was so irregular, that proper formation of the podosomes was prevented, leading to unstable and short‐lived podosomes. This was coupled to an impaired function of the podosomes, as the degradation potential of these podosome‐displaying cells was significantly reduced.

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Citation

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Chicago
De Clercq, Sarah. 2013. “Modulating L‐plastin Function in Immune Cells by Nanobodies : How Small Bodies Can Have Large Consequences”. Ghent, Belgium: Ghent University. Faculty of Medicine and Health Sciences.
APA
De Clercq, Sarah. (2013). Modulating L‐plastin function in immune cells by nanobodies : how small bodies can have large consequences. Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium.
Vancouver
1.
De Clercq S. Modulating L‐plastin function in immune cells by nanobodies : how small bodies can have large consequences. [Ghent, Belgium]: Ghent University. Faculty of Medicine and Health Sciences; 2013.
MLA
De Clercq, Sarah. “Modulating L‐plastin Function in Immune Cells by Nanobodies : How Small Bodies Can Have Large Consequences.” 2013 : n. pag. Print.
@phdthesis{4149990,
  abstract     = {The actin cytoskeleton is a crucial part of both eukaryotic and prokaryotic cells. This can be taken literally, as it is the largest cellular system found in cells. Furthermore, the cytoskeleton is also crucial for the correct functioning of the cell, since it is in charge of the shape, polarity, motility, division, differentiation, contraction, adhesion,{\textellipsis} of the cell. It goes without saying that, in order to fulfill this list of tasks, this structure has to be dynamic, always ready to adapt to the changing needs of the cell. However, as is often the case for rather cumbersome organelles, they rely on little, versatile players, to make them more dynamic. These are here represented by the so\unmatched{2010}called {\textquotedblleft}actin binding proteins{\textquotedblright} (ABPs), a plethora of actin polymerizing/depolymerizing, bundling/crosslinking, capping, monomer binding,{\textellipsis} proteins which regulate the above mentioned processes.
This thesis focuses (in descending order of importance) on the bundling protein L\unmatched{2010}plastin (LPL), the severing and capping protein gelsolin (GSN), and to a lesser extent on the capping protein CapG. Lplastin (or leukocyte plastin) is exclusively expressed in hematopoietic cells and ectopically in several cancer cells. Gelsolin and CapG are both abundantly expressed in most cell types. Nanobodies (the variable part of camelid heavy chain only antibodies) targeting these three ABPs have previously been characterized in our lab and have proven to be bona fide antagonists of these proteins in cancer cells, counteracting their motility and invasion. In this thesis, these nanobodies were employed in another cell context, namely in immune\unmatched{2010}related phenomena.
In a first part of this thesis, Nbs targeting L\unmatched{2010}plastin and gelsolin were used in a study concerning the formation of the immune synapse (IS) and subsequent T cell activation, an important event in the initiation of the immune response following insult by pathogens and toxins. L\unmatched{2010}plastin has been shown before to play a role in the formation of the IS between antigen presenting cells (APCs) and T cells. A possible role for gelsolin has not been investigated before. The use of Nbs, targeting distinct domains of L\unmatched{2010}plastin and gelsolin, makes it possible to reveal subtle mechanistic details as particular functions of these proteins can be silenced. One LPL Nb specifically interacts with the EF hands, thereby sequestering L\unmatched{2010}plastin in an inactive conformation, preventing it from interacting with actin. Another LPL Nb binds to the actin binding domains of L\unmatched{2010}plastin and interferes with actin bundling. When expressed in human T cells, both LPL Nbs perturbed IS formation as well as the polarization of the T cell toward the center of the APC, with subsequent docking of the microtubule organizing center (MTOC). Both Nbs also delayed L\unmatched{2010}plastin phosphorylation, which is required for optimal bundling. One Nb reduced the interaction between L\unmatched{2010}plastin and the integrin LFA\unmatched{2010}1, which is important for the adhesion between T cells and APCs. Interaction of L\unmatched{2010}plastin with LFA\unmatched{2010}1 is thought to stabilize LFA\unmatched{2010}1 at the immune synapse and indeed, LFA\unmatched{2010}1 enrichment at the synapse was reduced in the presence of this Nb. Furthermore, this Nb delayed Ser745 phosphorylation of LFA\unmatched{2010}1. Consequently, T cell activation was defective, as demonstrated by a hampered T cell proliferation and diminished interleukin\unmatched{2010}2 (IL\unmatched{2010}2) secretion. Although gelsolin was shown to be also present at the IS, a nanobody targeting the actin\unmatched{2010}free population of gelsolin had no effect on immune synapse formation and subsequent T cell activation.
In a second part of this thesis, L\unmatched{2010}plastin, gelsolin and CapG Nbs were employed in a study concerning podosomes. These are dynamic adhesion and degradation structures, consisting of an actin\unmatched{2010}rich core, surrounded by a ring of adhesion molecules. They are found in monocytic, smooth muscle and endothelial cells, as well as in transformed fibroblasts. L\unmatched{2010}plastin and gelsolin have repeatedly been described to colocalize with F\unmatched{2010}actin in the podosomal core, and are thought to fulfill important, yet distinct, roles in podosome assembly, turnover, stabilization and/or function. We further showed that, unlike the other 2 proteins, CapG is not enriched in podosomes of macrophage\unmatched{2010}like THP\unmatched{2010}1 cells. Also here, distinct nanobodies were used which revealed novel insights. A gelsolin nanobody specifically targeting active, actin\unmatched{2010}bound gelsolin, was enriched in podosomes. This was not the case for a nanobody targeting actin\unmatched{2010}free gelsolin. However, the presence of the above described L\unmatched{2010}plastin nanobodies had a serious impact on podosome formation and function. Cells expressing these Nbs were not capable anymore of forming stable podosomes, demonstrating the (normal) regular and cyclic turnover of Factin was lost. Instead, this turnover was so irregular, that proper formation of the podosomes was prevented, leading to unstable and short\unmatched{2010}lived podosomes. This was coupled to an impaired function of the podosomes, as the degradation potential of these podosome\unmatched{2010}displaying cells was significantly reduced.},
  author       = {De Clercq, Sarah},
  language     = {eng},
  pages        = {XII, 251},
  publisher    = {Ghent University. Faculty of Medicine and Health Sciences},
  school       = {Ghent University},
  title        = {Modulating L\unmatched{2010}plastin function in immune cells by nanobodies : how small bodies can have large consequences},
  year         = {2013},
}