The interaction between lipid derivatives of colchicine and tubulin: consequences of the interaction of the alkaloid with lipid membranes.

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin-water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed.

The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes.

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

Protein interactions in the calf eye lens: interactions between beta-crystallins are repulsive whereas in gamma-crystallins they are attractive.

Non-specific interactions in beta- and gamma-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7 degrees C and 31 degrees C. Two types of interactions were observed. With beta-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.

Molecular basis of eye lens transparency. Osmotic pressure and X-ray analysis of alpha-crystallin solutions.

Short range, liquid-like order of the crystallin proteins accounts for eye lens transparency. The relationship between structural and thermodynamic properties of eye lens was further investigated using osmotic pressure and small-angle X-ray scattering measurements of calf lens alpha-crystallins. The consistency of both data sets confirms that the macroscopic thermodynamic properties are determined by the structural properties accessible to X-ray scattering. In addition, the experimental data were correctly accounted for using a model developed in liquid-state physics: the rescaled mean spherical approximation combined with a Verwey-Overbeek potential. This model provides as best fit parameters the excluded volume, the charge and the diameter of an "equivalent" particle that compare well with the corresponding values found in the literature for alpha-crystallins. As a result, transparency may now be expressed as a function of a few structural parameters, the role of which is discussed. The approach presented here may be extended to studies of the thermodynamic-structural relationships of other protein solutions.

The protein concentration gradient within eye lens might originate from constant osmotic pressure coupled to differential interactive properties of crystallins.

A protein concentration gradient exists from the center to the periphery of most lenses, the origin of which is still a matter of debate. The gradient, which contributes to the lens optical quality, seems to be accompanied by an uneven distribution of the crystallin classes, with the nucleus usually enriched in gamma- and the cortex in alpha-crystallins. Since the osmotic pressure within the lens seems to be constant and since a rather different interaction behaviour of alpha- and gamma-crystallins was demonstrated in previous studies, we propose that the maintenance of a constant osmotic pressure through the lens is sufficient to induce and stabilize a protein concentration gradient. The theoretical treatment has been worked out and the validity of the hypothesis has been demonstrated with colloidal osmotic pressure measurements of lens cortical and nuclear cytoplasmic extracts as a function of protein concentration. To account for the observed lens concentration gradient, however, a small additional concentration gradient in the opposite direction, involving an ion or small molecule, might be required.