Multi-scale mechanical characterization of prostate cancer cell lines: Relevant biological markers to evaluate the cell metastatic potential.

BACKGROUND: Considering the importance of cellular mechanics in the birth and evolution of cancer towards increasingly aggressive stages, we compared nano-mechanical properties of non-tumoral (WPMY-1) and highly aggressive metastatic (PC-3) prostate cell lines both on cell aggregates, single cells, and membrane lipids. METHODS: Cell aggregate rheological properties were analyzed during dynamic compression stress performed on a homemade rheometer. Single cell visco-elasticity measurements were performed by Atomic Force Microscopy using a cantilever with round tip on surface-attached cells. At a molecular level, the lateral diffusion coefficient of total extracted lipids deposited as a Langmuir monolayer on an air-water interface was measured by the FRAP technique. RESULTS: At cellular pellet scale, and at single cell scale, PC-3 cells were less stiff, less viscous, and thus more prone to deformation than the WPMY-1 control. Interestingly, stress-relaxation curves indicated a two-step response, which we attributed to a differential response coming from two cell elements, successively stressed. Both responses are faster for PC-3 cells. At a molecular scale, the dynamics of the PC-3 lipid extracts are also faster than that of WPMY-1 lipid extracts. CONCLUSIONS: As the evolution of cancer towards increasingly aggressive stages is accompanied by alterations both in membrane composition and in cytoskeleton dynamical properties, we attribute differences in viscoelasticity between PC-3 and WPMY-1 cells to modifications of both elements. GENERAL SIGNIFICANCE: A decrease in stiffness and a less viscous behavior may be one of the diverse mechanisms that cancer cells adopt to cope with the various physiological conditions that they encounter.

Structural comparison of highly similar nucleoside-diphosphate kinases: Molecular explanation of distinct membrane-binding behavior.

NDPK-A, NDPK-B and NDPK-D are three enzymes which belong to the NDPK group I isoforms and are not only involved in metabolism process but also in transcriptional regulation, DNA cleavage, histidine protein kinase activity and metastasis development. Those enzymes were reported to bind to membranes either in mitochondria where NDPK-D influences cardiolipin lateral organization and is thought to be involved in apoptotic pathway or in cytosol where NDPK-A and NDPK-B membrane association was shown to influence several cellular processes like endocytosis, cellular adhesion, ion transport, etc. However, despite numerous studies, the role of NDPK-membrane association and the molecular details of the binding process are still elusive. In the present work, a comparative study of the three NDPK isoforms allowed us to show that although membrane binding is a common feature of these enzymes, mechanisms differ at the molecular scale. NDPK-A was not able to bind to model membranes mimicking the inner leaflet of plasma membrane, suggesting that its in vivo membrane association is mediated by a non-lipidic partner or other partners than the studied phospholipids. On the contrary, NDPK-B and NDPK-D were shown to bind efficiently to liposomes mimicking plasma membrane and mitochondrial inner membrane respectively but details of the binding mechanism differ between the two enzymes as NDPK-B binding necessarily involved an anionic phospholipid partner while NDPK-D can bind either zwitterionic or anionic phospholipids. Although sharing similar secondary structure and homohexameric quaternary arrangement, tryptophan fluorescence revealed fine disparities in NDPK tertiary structures. Interfacial behavior as well as ANS fluorescence showed further dissimilarities between NDPK isoforms, notably the presence of distinct accessible hydrophobic areas as well as different capacity to form Gibbs monolayers related to their surface activity properties. Those distinct features may contribute to explain the differences in the protein behavior towards membrane binding.

New insights into lipid-Nucleoside Diphosphate Kinase-D interaction mechanism: protein structural changes and membrane reorganisation.

Nucleoside Diphosphate Kinases (NDPKs) have long been considered merely as housekeeping enzymes. The discovery of the NME1 gene, an anti-metastatic gene coding for NDPK-A, led the scientific community to re-evaluate their role in the cell. It is now well established that the NDPK family is more complex than what was first thought, and despite the increasing amount of evidence suggesting the multifunctional role of nm23/NDPKs, the specific functions of each family member are still elusive. Among these isoforms, NDPK-D is the only one to present a mitochondria-targeting sequence. It has recently been shown that this protein is able to bind and cross-link with mitochondrial membranes, suggesting that NDPK-D can mediate contact sites and contributes to the mitochondrial intermembrane space structuring. To better understand the influence of NDPK-D on mitochondrial lipid organisation, we analysed its behaviour in different lipid environments. We found that NDPK-D not only interacts with CL or anionic lipids, but is also able to bind in a non negligible manner to zwitterionic PC. NDPK-D alters membrane organisation in terms of fluidity, hydration and lipid clustering, effects which depend on lipid structure. Changes in the protein structure after lipid binding were evidenced, both by fluorescence and infrared spectroscopy, regardless of membrane composition. Taking into account all these elements, a putative mechanism of interaction between NDPK-D and zwitterionic or anionic lipids was proposed.

Mitochondrial creatine kinase binding to phospholipids decreases fluidity of membranes and promotes new lipid-induced beta structures as monitored by red edge excitation shift, laurdan fluorescence, and FTIR.

Structural modifications induced by the binding of mitochondrial creatine kinase (mtCK) to saturated and unsaturated phospholipids were monitored by using Laurdan, a membrane probe sensitive to the polarity of the environment. The abrupt change characteristic of a phase transition of lipids alone was attenuated by addition of mtCK. Generalized polarization spectra indicated that mtCK surface binding changed the phospholipid liquid-crystalline state to a more rigid state. Infrared spectra of lipids further strengthened these results: upon mtCK binding, the phospholipid methylene chains had a more rigid conformation than that observed without mtCK at the same temperature. After mtCK binding to vesicles of perdeuterated dimyristoylphosphatidylcholine and nondeuterated dimyristoylphosphatidylglycerol, no lateral phase separation was observed, suggesting that both lipids were rigidified. Moreover, mtCK bound to liposomes exhibited an uncommon red edge excitation shift of 19 nm, while that of the soluble enzyme was only 6 nm. These results indicated that the environment of some mtCK tryptophan residues was motionally restricted. Strong stabilization of the enzyme structure against heat denaturation was observed upon lipid binding. In addition, lipids promoted a new reversible protein-protein or protein-lipid interaction, as evidenced by infrared data showing a slight modification of the beta sheet over alpha helix ratio with formation of a new 1632-cm(-)(1) beta sheet instead of the soluble protein 1636-cm(-)(1) one. Such modifications, inducing a decrease in the fluidity of the mitochondrial membranes, may play a role in vesicle aggregation; they could be implicated in the appearance of contact sites between internal and external mitochondrial membranes.

Structural changes of mitochondrial creatine kinase upon binding of ADP, ATP, or Pi, observed by reaction-induced infrared difference spectra.

Structural modifications of rabbit heart mitochondrial creatine kinase induced by the binding of its nucleotide substrates and Pi were investigated. Reaction-induced difference spectra (RIDS), resulting from the difference between infrared spectra recorded before and after the photorelease of a caged ligand, allow us to detect very small variations in protein structure. Our results indicated that the protein secondary structure remained relatively stable during nucleotide binding. Indeed, this binding to creatine kinase affected only a few amino acids, and caused small peptide backbone deformations and alterations of the carbonyl side chains of aspartate or glutamate, reflecting modifications within preexisting elements rather than a net change in secondary structure. Nonetheless, MgADP and MgATP RIDS were distinct, whereas the MgPi RIDS presented some similarities with the MgATP one. The difference between MgADP and MgATP RIDS could reflect a distinct configuration of the two metal-nucleotide complexes inducing a different positioning and/or a distinct binding mode to the creatine kinase active site. Comparison of the MgATP and MgPi RIDS suggests that Pi binding took place at the same binding site as the gamma-phosphoryl group of ATP. Thus, the difference between MgADP and MgATP RIDS would mainly be due to the effect of the gamma-P of ATP. The differences observed when comparing the RIDS resulting from the binding of nucleotides to octameric mitochondrial creatine kinase or dimeric cytosolic isoform could reflect the distinct oligomerization states and physicochemical or kinetic properties of the two isoenzymes.

Mitochondrial creatine kinase binding to liposomes and vesicle aggregation: effect of cleavage by proteinase K.

Mitochondrial creatine kinase and its proteinase K nicked-derivative interaction with liposomes induced slight secondary structure changes evidenced by infrared spectra. In nondenaturing conditions, the N-terminal (K1) and the C-terminal (K2) fragments remained associated with each other and bound to liposomes. When the two fragments were separated by denaturation, K2 was soluble, whereas most of K1 was adsorbed onto liposomes. The three-dimensional structure of uncleaved mtCK suggests that the C-terminal moiety, which contains positively charged surface residues, interacted with membranes. After denaturation and renaturation of the nicked enzyme, both peptides did not refold properly and did not reassociate with each other. The misfolded K1 fragment bound to the membrane through a stretch of positive residues, which were buried in the native enzyme. The lack of binding of the ill-folded K2 peptide could be related to the disruption of the optimal disposition of its positive charges, responsible for the correct interaction of native mtCK with membrane.

Cloning, Escherichia coli expression, and phase-transition chromatography-based purification of recombinant rabbit heart mitochondrial creatine kinase.

A cDNA clone of the mitochondrial sarcomeric creatine kinase cDNA was obtained by screening a rabbit heart library. This cDNA is characterized by a 1257-nucleotide open reading frame encoding a 419-amino-acid protein with a cleavable 39-amino-acid mitochondrial presequence (Accession No. AJ011334). This new member of the guanidino kinase family shows a high degree of sequence similarity with the other phosphagen kinases sequenced so far. The mature enzyme was efficiently expressed in Escherichia coli BL21(DE3) cells as a soluble octameric protein using the pET21 plasmid and purified by a three-step improved method including a final phase-transition chromatography.