Physics of the cytoskeleton and membrane functions

Keywords: cytoskeleton dynamics, cell motility, epithelium, tumorigenesis, spectroscopy, melanin, melanoma

Dynamic control of intracellular processes

Deciphering cell processes mostly consists in studying structure-function relations. This makes it difficult to take into account the fact that living objects are out of equilibrium. With cytoskeleton physicochemistry as a starting point, our team has worked, since its inception, on the quantitative exploration of the cytoskeleton dynamics in play in cell self-assembly and maintenance of cell architecture. In order to better understand the relationships between dynamics and function, our current work focuses more specifically on the role of dynamics in two opposed phenomena: a dynamic instability necessary for cell movement and the dynamic stabilization of epithelia.

Movie 1: The amoeba entamoeba histolytica is filmed under phase contrast microscopy. It produces membrane protrusions, caused by abrupt detachment of the membrane from the underlying cytoskeleton, under the effect of excessive internal pressure resulting from permanent actin cortex contraction. Membrane detachment then leads to local cytoskeleton collapse, to the formation of new cortex that stabilizes, then finally to the cyclic repeat of all steps. This cortex instability is necessary for amoeba motility and infectivity.

We are approaching these questions thanks to the multi-disciplinary nature of our team - physicists, biologists and physicians - and using techniques such as cell and molecular biology, imaging, spectroscopy, micromanipulation, photomanipulation and modelling.

On the one hand, we are studying so-called "amoeboid" cell motility, which is the result of a finely controlled instability of the cytoskeleton associated with the plasma membrane. Though a combination of experimentation and theory, we study the physical mechanism of this dynamic instability, thus hoping to simplify the study of the control pathways of this type of cell motility involved in the migration of cancer cells through "soft" tissue. We are also attempting to understand how cancer or immunocompetent cells choose their the migration mode in 3D, amoeboid or mesenchymal.

On the other hand, because the mechanical stability between epithelial cells requires the balance of the intercellular forces transmitted via cell junctions, we have postulated that force balance between adjacent cells cannot be achieved statically, but that it requires permanent correction of imbalanced intracellular forces. This assumption confers a central role to cytoskeleton dynamics and to intercellular adhesion structures in the homeostasis of tissue geometry. It also implies a mechanosensory control of cytoskeleton renewal processes. Thus, the histological disorder observed during the initial stages of tumour transformation could result from abnormal stabilization dynamics. The first phase of our work focused on the dynamics of E-cadherin renewal, the role of endocytosis and the link between tissue dynamics and stability.

Fig. 1 

Our results demonstrate a clear correlation between epithelial destabilization mediated by the oncogene c-Met and faster cadherin dynamics. Endocytosis, a process required for the dissociation of adhesive interactions - probably under mechanosensory control - appears to play a key role. Based on these results, we wish to quantify histological disorder and to study the response junctions to asymmetric destabilization. We also intend to more generally study the propagation of the tumour phenotype, or phenotypic suppression induce by local oncogene activation.

Melanin spectroscopy and its medical applications

Multi-photon optical microscopy, in use by the team since its creation, is now widely provided to our colleagues through a large number of collaborations.

Fig. 2 

This tool has enabled us to observe an unexpected property of melanosomes, skin pigments made of condensed solid-state melanin. When excited by a femtosecond laser source, melanosomes display an intensity threshold beyond which their dim fluorescence is dominated by a very intense luminescence. No such threshold effect has been reported for biomolecules. Since melanin is a semiconductor, we studied the physics of this phenomenon, with the hypothesis of an optical transition similar to a micro-laser. Using several spectroscopy tools - spectral and temporal analysis, pulse train formatting - we discovered that this luminescence originates from a thermal effect, due to the sp2 carbon-rich structure of melanosomes. We further investigate the fundamental properties of this radiation, particularly in the near infrared field, but we already started to work towards the clinical application of this discovery. Indeed, photo-induced melanosome luminescence is both intense and highly specific and we wish to use it as a new signature for imaging surgical excision margins of melanoma. We also plan to use it for detecting and caracterizing melanoma metastasis-carrying cells in the peripheral blood. Thanks to the presence of a dermatolo-oncologist in our team, this work is being conducted in close collaboration with the hospital.

Prospects

We wish to further link our fundamental research lines with basic medical problems in the field of oncology, as well as with diagnostic applications. We are further studying melanosomes spectroscopy, along with its applications to circulating cells and microscopy. In imaging, the clinical validation of confocal reflectance microscopy for the diagnosis of melanoma led us to propose specialist consultations for high-risk patients and we will collaborate with the surgery department, to validate laser scanning macro/microscopic imaging for checking tumor excision margins.

Our fundamental work on the origin of epithelial stability and dysplasic disorder has raised a central question: to what extent do genetic causes currently associated with tumour transformation - oncogene activation for example - lead, or not, to tumour development when locally introduced into one or more cells, in an initially intact cell environment? This question, related to the notion of phenotypic suppression, leads us to study the collective effects of mutual control amongst cells within a tissue. Using phase transition concepts and tools at our disposal - 3D microfabrication, local oncogene photoactivation, the quantification of histological disorder - we hope to gain an understanding of tumor growth nucleates and expands within a group of cells.

Last update: February 2009

Key publications

2009

• Pellacani G, Longo C, Ferrara G, Cesinaro AM, Bassoli S, Guitera P, Menzies SW, Seidenari S.
  Spitz nevi: In vivo confocal microscopic features, dermatoscopic aspects, histopathologic correlates, and diagnostic significance
  J. Am. Acad. Dermatol., 60, 236-47
• Guitera P, Pellacani G, Longo C, Seidenari S, Avramidis M and Menzies SW
  In vivo reflectance confocal microscopy enhances secondary evaluation of melanocytic lesions
  J. Invest. Dermatol., 129, 131-8
• Mittelbrunn M, Martinez del Hoyo G, Lopez-Bravo M, Martin-Cofreces N B, Scholer A, Hugues S, Fetler L, Amigorena S, Ardavin C, Sanchez-Madrid F
  Imaging of plasmacytoid dendritic cell interactions with T cells
  Blood, 113(1):75-84
• Boissonnas A, Scholer-Dahirel A, Fetler L, and Amigorena S
  Multiphoton imaging of cytotoxic T lymphocyte-mediated anti-tumor immune reponses
  Curr. Top. Microbiol. Immunol., 334, 265-287
• De Beco S, Gueudry C, Amblard F, Coscoy S
  Endocytosis is required for E-cadherin redistribution at mature adherens junctions
  PNAS April 28, vol. 106 no. 17 7010-7015

2008

• Guitera P, Li LX, Crotty K, Fitzgerald P, Mellenbergh R, Pellacani G, Menzies SW.
  Melanoma histological Breslow thickness predicted by 75-MHz ultrasonography
  Br. J. Dermatol., 159, 364-9
• Pulecio J, Tagliani E, Scholer A, Prete F, Fetler L, Burrone O R, Benvenuti F
  Expression of Wiskott-Aldrich Syndrome Protein in Dendritic Cells Regulates Synapse Formation and Activation of Naive CD8+ T Cells
  J Immunol., 181, 1135-42
• Scholer A., et al.
  Intercellular adhesion molecule-1-dependent stable interactions between T cells and dendritic cells determine CD8⁺ T cell memory
  Immunity, 28, 258-270

2007

• Pellacani G, Guitera P, Longo C, Avramidis M, Seidenari S, Menzies SW
  The impact of in vivo reflectance confocal microscopy for the diagnostic accuracy of melanoma and equivocal melanocytic lesions
  J. Invest. Dermatol., 127, 2759-65
• C. Gueudry, et al.
  Protein dynamics in living cells: multidimensional imaging, Photo perturbation and automated analysis in modern life microscopy
  Imaging & Microscopy, 8: 24-26