Research Interests: John Robertson

Cellular Membrane Structure and Function
Animal Physiology, Compartative Physiology, Environmental Physiology, Fish Physiology
Adaptation and Acclimation

Cholesterol, Plasma Membranes and Temperature Acclimation
The body temperature of poikilotherms, including most fish, varies with environmental temperature. Homeoviscous theory holds that compensatory restructuring of cellular membrane lipid composition is central to successful temperature acclimation. In this way, vital membrane physical properties ('fluidity'), and associated membrane functions (e.g., acting as diffusion barriers or matrices for protein activity) can be maintained in a physiologically permissive range. To investigate the role cholesterol plays in thermally-induced membrane restructuring, I isolated plasma membranes from several trout tissues and quantified cholesterol content (1). The elevated cholesterol content seen with acclimation to the warmer temperature is consistent with biophysical studies indicating that cholesterol orders most bilayer membranes; thus, incorporation of stabilizing cholesterol could offset the molecular disorder introduced into the membrane by higher temperature (2). One particularly intriguing aspect of these data is the high cholesterol levels in gill plasma membranes relative to other tissues. This result provoked pursuit of my current primary research interest: does cell membrane lipid composition influence the functional properties of fish gills?
Fish Gill Barrier Membranes, Cholesterol and Water Permeability
Given their range of homeostatic functions and interfacial disposition with the ambient environment, gills are unique among physiologically active epithelia. Morphologically, gills feature amplified exchange surface areas and minimal blood-to-water diffusion distances; characteristics thought functionally important for, inter alia, oxygen uptake. However, in freshwater fish these structural attributes come at a potentially significant cost: increased osmotic water uptake (a situation that has been termed the 'osmorespiratory compromise'). Especially since the discovery of the aquaporins (protein water channels), the concept that specific lipid composition could limit the water permeability of biological membranes has gained increasing attention. Cholesterol is well known to limit transmembrane permeabilities in model systems; therefore, I am very interested in the relationship between membrane lipid composition (particularly cholesterol) and water permeability in the barrier membranes of gills of freshwater fish. I have used isolated ligated gill arches from trout and warmwater tilapia to demonstrate that osmotic water uptake is both temperature dependant and influenced by
acclimation status; properties consistent with a lipid bilayer water permeation pathway (3). Moreover, using isolated arches, I have gathered evidence for the importance cholesterol plays in restricting gill water permeability by: a) using a cholesterol-complexing, pore-forming antibiotic (nystatin) to increase osmotic water uptake, and: b) employing cyclodextrin (CD) to manipulate the cholesterol level, and water uptake rates, of gill barrier membranes. I have also been using another cholesterol-complexing antibiotic, filipin, as a fluorescent cytochemical marker for cholesterol in gill surface membranes (right).
     These studies represent a foundation for pursuing more direct, molecular-level investigations of membrane structure/function relationships; e.g., isolating purified gill barrier membranes and characterizing their lipid composition, membrane physical properties and permeability characteristics. Ultimately, isolated gill barrier membranes represent a highly relevant biological model for exploring a variety of basic and applied physiological questions. Examples range from the effects of temperature, salinity and xenobiotics on gill interfacial membrane composition and functional properties to the issue of whether water and oxygen permeability are independently influenced by membrane lipid composition. I am also interested in whether certain anthropogenic (e.g., icthyotoxins containing saponins) and natural (algal) toxins interact in a lipid composition dependent manner with gill barrier membranes.
Gill Pillar Cells, Ectoenzymes and Signal Transduction
In characterizing trout plasma membranes, the activities of two plasma membrane enzymes, 5'nucleotidase (5'NT) and alkaline phosphodiesterase (APD), were found to be elevated in tissue homogenates and isolated plasma membranes from gills relative to other tissues. These enzymes are widely used as markers for the apical plasma membrane domain of vertebrate epithelial cells. My interest in isolating gill epithelial cell apical plasma (barrier) membranes lead me to further characterize these enzymes in the gill. I used histochemistry to localize both 5'NT and APD activities exclusively to the pillar cells which form the central microvascular network in the gill epithelial outfoldings or lamellae (4). Significantly, there is no indication of either enzyme activity associated with the surface gill epithelial cells. These results will be quite valuable for demonstrating effective separation of vascular and epithelial plasma membranes when isolating gill barrier membranes. Histochemical staining patterns were consistent with an apical plasma membrane localization for both activities in pillar cells - i.e., the enzyme activities were found at the membranes forming the actual microvascular lining. The functional significance of this localization is intriguing. Both 5'NT and APD are involved in nucleotide metabolism and have been suggested to act as modulators in purinergic signal transduction, and pillar cells are unique microvascular cells with apparent endogenous contractile capacity. Could these enzyme activities reflect a mechanism for local regulation of hemodynamics in gill lamellae, perhaps in response to hypoxia or other environmental conditions? The same pillar cell localization for 5'NT and APD has been seen in all species of fish examined (freshwater trout and tilapia as well as seawater flounder and eel), suggesting that any associated physiological functions may be broadly conserved. I plan to pursue functional questions and continue structural investigations of gill pillar cells using immunolocalization and biochemical approaches.
Developmental Biology, Sex Differentiation, Smoltification
Interests in developmental biology and endocrine physiology derive from work on the ontogeny of a sexual dimorphism in axon numbers in the laryngeal neuromuscular system of Xenopus (5). One project initiated in these studies examined primary (gonadal) versus secondary (laryngeal masculinization) sexual development in tadpoles. By chemically arresting metamorphosis, we demonstrated that while the gonads differentiate independent of thyroid hormone (TH), the ability of the larynx to be masculinized by androgens depends upon exposure to TH (6). I would like to return to this system to look specifically at cellular and molecular hallmarks of maturation in the developing gametes of metamorphically blocked tadpoles. This work is provocative from an evolutionary perspective in that it may illuminate a mechanistic basis for neoteny - a phenomenon in some amphibian species whereby larval forms become reproductively mature.
     I also have background and interests in more applied aspects of fish biology stemming from my experiences with aquaculture as an extension agent in the Peace Corps and aspects of my master's degree work - involving, for example, environmental influences on smoltification (fresh- to seawater transformation) in anadromous salmonids (7).

(1) Robertson, J.C. and Hazel, J.R. 1995. Cholesterol content of trout plasma membranes varies with acclimation temperature. American Journal of Physiology, 269: R1113-R1119.
(2) Robertson, J.C. and Hazel, J.R. 1997. Membrane constraints to physiological function at different temperatures: Does cholesterol stabilize membranes at elevated temperatures? In: Global Warming - Implications for Freshwater and Marine Fish. Eds. C. Wood and G. McDonald. pp. 25-49. Society for Experimental Biology Seminar Series. 
(3) Robertson, J.C. and Hazel, J.R. 1999. Influence of temperature and membrane lipid composition on the osmotic water permeability of teleost gills. Physiological and Biochemical Zoology. 72: 623-632.
(4) Robertson, J.C. and Hazel, J.R. 1998. 5'Nucleotidase and alkaline phosphodiesterase activities in trout gill localize to endothelial (pillar) cells. Journal of Experimental Biology, 201: 2011-2019. 
(5) Robertson, J., Watson, J. and Kelley, D. 1994. Androgen directs sexual differentiation of laryngeal innervation in developing Xenopus laevis. Journal of Neurobiology, 25: 1625-1636. 
(6) Robertson, J.C. and Kelley, D.B.1996. Thyroid hormone controls the onset of androgen sensitivity in the developing larynx of Xenopus laevis. Developmental Biology, 176: 108-123.
(7) Robertson, J.C. and Bradley, T.M. 1991. Hepatic ultrastructure changes associated with the parr-smolt transformation of Atlantic salmon (Salmo salar ). Journal of Experimental Zoology, 260: 135-148.