Melanophores : cell biophysics and sensor applications

Abstract: This thesis deals with studies of melanophores and melanosomes by means of a physical approach. Melanophores are pigment cells that give the black colour to many vertebrates, e.g. fishes, frogs, and reptiles. Covering large areas of the skin, these cells are approximately 0.1 mm in diameter, and they contain black pigment granules called melanosomes.The geometry and the electric charge of isolated melanosomes were two physical properties that were studied. The electric charge was measured by electrophoresis and was found to be (-1.7 ± 0.2)·10-16 Coulomb in average. The geometry of melanosomes was measured using Scanning Force Microscopy, SFM, and resulted in an ellipsoidal shape with an average major diameter of 840 ± 20 nm.Under nervous and hormonal control, melanophores rearrange the intracellular melanosomes from a scattered distribution, called dispersed, to a state where all melanosomes are accumulated in the cell centre, called the aggregated state. In this way, melanophores change from black towards transparent. This gives an animal the ability to change not only between being pale or dark, but also between different colours by using melanophores to cover and uncover the colours of different types of pigment cells from underlying layers.The volume of melanosomes was measured with SFM. This study resulted in a difference of 18% when individual melanosomes from aggregated and dispersed melanophores were measured separately.Magnetic field exposure of melanophores has been reported to affect the aggregation. However, contradicting results are presented in the literature. To clarify the possible effect of magnetic fields on melanophores, experiments by aggregating fish melanophores under exposure to strong (8 and 14 Tesla), homogenous, static magnetic fields were carried out. Both the magnetic field-induced Lorentz force on the charged melanosomes and the reorientation of the cytoskeleton were considered as possible explanations of any effects. Whenfield experiments were compared to control experiments with zero field, no difference in aggregation levels were found. However, a more irregular speed of aggregation was seen in the 8 Tesla field than in the control experiments.A theoretical model was developed to explain switch-like responses in biological systems. A switch-like response to a graded stimulation was sometimes seen in the case of melanophores but was shown not to have a very large so-called Hill coefficient. The model is simple in its approach. It may be applied to general phenomena and is based on the assumption of a simultaneous desorption of an activator (agonist; substrate molecule; ... ) and an inactivator (antagonist, inhibitor; ... ) caused by a collision or interaction between two effector molecules (e.g. receptors or enzymes).Melanosomes are also found in the human body and have a remarkably capacity to bind other molecules. A well-established forensic application of this is to detect (illegal) drugs that have bound to melanosomes in hair shafts. So far this application is only qualitative. This thesis includes a characterisation of the binding of flunitrazepam to melanin. Flunitrazepam is the active substance of Rohypnol, which is a sedative that is illegal in several countries and sometimes called the "date-rape-drug".Melanophores are excellent model systems for studies of cellular phenomena. Moreover, melanophores are also interesting in sensing aspects. The change from black to transparent is a highly visible response to substances in their surroundings and has previously been the measurand in melanophore-based biosensors. The physical approach of these studies of melanophores also had the objective of evaluating new biosensor solutions.