The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

We describe a structural domain common to proteins related to human calmodulin-regulated spectrin-associated protein1 (CAMSAP1). Analysis of the sequence of CAMSAP1 identified a domain near the C-terminus common to CAMSAP1 and two other mammalian proteins KIAA1078 and KIAA1543, which we term a CKK domain. This domain was also present in invertebrate CAMSAP1 homologues and was found in all available eumetazoan genomes (including cnidaria), but not in the placozoan Trichoplax adherens, nor in any nonmetazoan organism. Analysis of codon alignments by the sitewise likelihood ratio method gave evidence for strong purifying selection on all codons of mammalian CKK domains, potentially indicating conserved function. Interestingly, the Drosophila homologue of the CAMSAP family is encoded by the ssp4 gene, which is required for normal formation of mitotic spindles. To investigate function of the CKK domain, human CAMSAP1-enhanced green fluorescent protein (EGFP) and fragments including the CKK domain were expressed in HeLa cells. Both whole CAMSAP1 and the CKK domain showed localization coincident with microtubules. In vitro, both whole CAMSAP1-glutathione-s-transferase (GST) and CKK-GST bound to microtubules. Immunofluorescence using anti-CAMSAP1 antibodies on cerebellar granule neurons revealed a microtubule pattern. Overexpression of the CKK domain in PC12 cells blocked production of neurites, a process that requires microtubule function. We conclude that the CKK domain binds microtubules and represents a domain that evolved with the metazoa.

The cells in animals face unique demands beyond those encountered by their unicellular eukaryotic ancestors. For example, the forces engendered by the movement of animals places stresses on membranes of a different nature than those confronting free-living cells. The integration of cells into tissues, as well as the integration of tissue function into whole animal physiology, requires specialisation of membrane domains and the formation of signalling complexes. With the evolution of mammals, the specialisation of cell types has been taken to an extreme with the advent of the non-nucleated mammalian red blood cell. These and other adaptations to animal life seem to require four proteins--spectrin, ankyrin, 4.1 and adducin--which emerged during eumetazoan evolution. Spectrin, an actin cross-linking protein, was probably the earliest of these, with ankyrin, adducin and 4.1 only appearing as tissues evolved. The interaction of spectrin with ankyrin is probably a prerequisite for the formation of tissues; only with the advent of vertebrates did 4.1 acquires the ability to bind spectrin and actin. The latter activity seems to allow the spectrin complex to regulate the cell surface accumulation of a wide variety of proteins. Functionally, the spectrin-ankyrin-4.1-adducin complex is implicated in the formation of apical and basolateral domains, in aspects of membrane trafficking, in assembly of certain signalling and cell adhesion complexes and in providing stability to otherwise mechanically fragile cell membranes. Defects in this complex are manifest in a variety of hereditary diseases, including deafness, cardiac arrhythmia, spinocerebellar ataxia, as well as hereditary haemolytic anaemias. Some of these proteins also function as tumor suppressors. The spectrin-ankyrin-4.1-adducin complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.

Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and differential mRNA splicing. Finally, the spectrum of interactions of the 4.1 proteins overlaps with that of another membrane-cytoskeleton linker, ankyrin. Both ankyrin and 4.1 link to the actin cytoskeleton via spectrin, and we hypothesize that differential regulation of 4.1 proteins and ankyrins allows highly selective control of cell surface protein accumulation and, hence, function. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.