Membrane adhesion and the formation of heterogeneities: biology, biophysics, and biotechnology.

Membrane adhesion is essential to many vital biological processes. Sites of membrane adhesion are often associated with heterogeneities in the lipid and protein composition of the membrane. These heterogeneities are thought to play functional roles by facilitating interactions between proteins. However, the causal links between membrane adhesion and membrane heterogeneities are not known. Here we survey the state of the field and indicate what we think are understudied areas ripe for development.

Cytokinesis failure in clathrin-minus cells is caused by cleavage furrow instability.

The role of membrane traffic during cell division has only recently begun to be investigated. A growing number of trafficking proteins seem to be involved in the successful completion of cytokinesis. Clathrin was the first trafficking protein to be shown to be essential for cytokinesis in Dictyostelium. Here we investigate the nature of the cytokinesis defect of Dictyostelium clathrin null cells. We found that adherent clathrin null cells do form cleavage furrows but cannot maintain a consistent rate of furrow ingression. Clathrin null cells are completely defective in cytokinesis when placed in suspension. In these conditions, the cells develop an abnormal division morphology that consists of two lateral "furrows" on either side of a bulging equatorial region. Cells expressing GFP-myosin II were examined at various stages of cytokinesis. Clathrin null cells show multiple defects in myosin organization and localization that parallel the striking failure in furrow morphology. We postulate that this morphology is the result of contraction at the rear of the presumptive daughter cells in concert with incomplete furrow ingression.

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation.

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.

Membrane traffic and cytokinesis.

Cytokinesis, the last step in cell division, is a process common to all eukaryotic life forms. The many mechanisms cells use to divide one parent cell into two progeny reflect the diversity of eukaryotic life. Despite the varied mechanisms cells use, increasing evidence demonstrates that many different cells use 'classical' membrane trafficking proteins for cytokinesis. This review highlights recent evidence for roles for membrane trafficking proteins in cytokinesis.

Clathrin plays a novel role in the regulation of cell polarity, pseudopod formation, uropod stability and motility in Dictyostelium.

Although the traditional role of clathrin has been in vesicle trafficking and the internalization of receptors, a novel role in cytokinesis was recently revealed in an analysis of a clathrin-minus Dictyostelium mutant (chc(-)). chc(-) cells grown in suspension were demonstrated to be defective in assembling myosin II into a normal contractile ring. To test whether this defect reflected a more general one of cytoskeletal dysfunction, chc(-) cells were analyzed for cell polarity, pseudopod formation, uropod stability, cell locomotion, chemotaxis, cytoskeletal organization and vesicle movement. chc(-) cells crawled, chemotaxed, localized F-actin in pseudopods, organized their microtubule cytoskeleton in a relatively normal fashion and exhibited normal vesicle dynamics. Although chc(-) cells extended pseudopods from the anterior half of the cell with the same frequency as normal chc(+) cells, they extended pseudopods at twice the normal frequency from the posterior half of the cell. The uropods of chc(-) cells also exhibited spatial instability. These defects resulted in an increase in roundness, a reduction in polarity, a reduction in velocity, a dramatic increase in turning, a high frequency of 180 degrees direction reversals and a decrease in the efficiency of chemotaxis. All defects were reversed in a rescued strain. These results are the first to suggest a novel role for clathrin in cell polarity, pseudopod formation, uropod stability and locomotion. It is hypothesized that clathrin functions to suppress pseudopod formation and to stabilize the uropod in the posterior half of a crawling cell, two behavioral characteristics that are essential for the maintenance of cellular polarity, efficient locomotion and efficient chemotaxis.

Cloning and characterization of a Dictyostelium gene encoding a small GTPase of the Rab11 family.

Eukaryotic cells achieve complexity by compartmentalizing a subset of cellular functions into membrane-bound organelles. Maintaining this high level of cellular organization requires precise regulation of traffic between membranes. This task is accomplished, in part, by rab proteins. How these small GTPases regulate membrane traffic between cellular compartments is not clear. Here we report the characterization of a novel rab GTPase from the soil amoebae Dictyostelium discoideum. The predicted coding sequence of the new rab gene, Dictyostelium rab11b, encodes a protein of 25 kD containing all the structural hallmarks of a rab GTPase. Comparison of the sequence with the GenBank database and cladistic analysis demonstrated Dictyostelium rab11b to be a divergent member of the rab11 branch of rab proteins. Southern analysis revealed the presence of related genes in Dictyostelium. RNAse protection assays showed the Dictyostelium rab11b gene to be expressed at uniform levels throughout growth and development. Gene deletion experiments revealed that Dictyostelium rab11b was not essential for growth or development. Conceivably, the function of rab11b may be redundant with that of related genes in this organism.

A novel role for clathrin in cytokinesis.

Using clathrin-minus Dictyostelium cells, we identified a novel requirement for clathrin during cytokinesis. In suspension culture, clathrin-minus cells failed to divide and became large and multinucleate. This cytokinesis deficiency was not attributable to a pleiotropic effect on the actomyosin cytoskeleton, since other cellular events driven by myosin II (e.g., cortical contraction and capping of concanavalin A receptors) remained intact in clathrin-minus cells. Examination of cells expressing myosin II tagged with green fluorescent protein showed that clathrin-minus cells failed to assemble myosin II into a functional contractile ring. This inability to localize myosin II to a particular location was specific for cytokinesis, since clathrin-minus cells moving across a substrate localized myosin II properly to their posterior cortexes. These results demonstrate that clathrin is essential for construction of a functional contractile ring during cell division.

Clathrin heavy chain is required for spore cell but not stalk cell differentiation in Dictyostelium discoideum.

Previous studies of a clathrin-minus Dictyostelium cell line revealed important roles for clathrin heavy chain (clathrin) in endocytosis, secretion of lysosomal hydrolases and osmoregulation. In this paper, we examine the contribution of clathrin-mediated membrane traffic to development in Dictyostelium discoideum. Clathrin-minus cells were delayed in early development. When exposed to starvation conditions, clathrin-minus cells streamed and aggregated more slowly than wild-type cells. Although clathrin-minus cells displayed only 40% the level of extracellular cyclic AMP binding normally found in wild-type cells, they responded chemotactically to extracellular cyclic AMP. Clathrin-minus cells down-regulated cyclic AMP receptors, but only to half the extent of wild-type cells. We found that the extent of development of clathrin-minus cells was variable and influenced by environmental conditions. Although the mutant cells always progressed beyond the tipped mound stage, the final structure varied from a finger-like projection to a short, irregular fruiting body. Microscopic examination of these terminal structures revealed the presence of intact stalks but a complete absence of spores. Clathrin-minus cells expressed prestalk (ecmA and ecmB) and prespore (psA and cotB) genes normally, but were blocked in expression of the sporulation gene spiA. Using clathrin-minus cells that had been transformed with various promoter-lacZ reporter constructs, we saw only partial sorting of clathrin-minus prestalk and prespore cells. Even when mixed with wild-type cells, clathrin-minus cells failed to sort correctly and never constructed functional spores. These results suggest three roles for clathrin during Dictyostelium development. First, clathrin increases the efficiency of early development. Second, clathrin enables proper and efficient patterning of prestalk and prespore cells during culmination. Third, clathrin is essential for differentiation of mature spore cells.

Purification of clathrin heavy and light chain from Dictyostelium discoideum.

Clathrin, a protein important for endocytosis, is a hexamer composed of three heavy chains and three light chains. We report here the purification scheme used to isolate the clathrin protein from the simple eukaryote, Dictyostelium discoideum. Using a combination of differential centrifugation and column chromatography, we isolated approximately 2 mg of clathrin triskelions from 150-200 g of Dictyostelium cells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine an S value of 7.9 for purified clathrin. Rotary shadowed images of Dictyostelium clathrin revealed trimeric molecules with extended legs measuring 48 +/- 5 nm, similar in length to the legs of mammalian and yeast clathrin triskelions. The single clathrin light chain proved resistant to heat treatment, a property also similar to light chains from other species. The conservation of these physical properties in Dictyostelium clathrin demonstrates the potential of this model organism for the study of clathrin structure and function.

Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium.

To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.

Characterization of the clathrin heavy chain from Dictyostelium discoideum.

We report the cloning and analysis of a clathrin heavy-chain cDNA from the eukaryotic microorganism, Dictyostelium discoideum. A single gene, designated chcA, for the clathrin heavy chain encoded a protein of 1,694 amino acids with a molecular mass of 193,618 daltons. Comparison of the amino acid sequence with the rat and with the yeast sequence showed that the highly conserved protein was more similar to the mammalian clathrin heavy chain (57% identity) than to the yeast heavy chain (45% identity). The mRNA for the clathrin heavy chain was regulated during development. mRNA levels were highest during vegetative growth and declined as the cells progressed through the 24-hr developmental cycle. The concentration of clathrin heavy-chain protein was the same in cells grown in liquid media (high rates of pinocytosis) as in cells grown with bacteria (low rates of pinocytosis), which suggests that regulation of pinocytosis in these cells is not achieved by altering the concentration of clathrin.

Genetically engineered truncated myosin in Dictyostelium: the carboxyl-terminal regulatory domain is not required for the developmental cycle.

The study of engineered Dictyostelium mutants with altered or missing myosin has revealed the molecule to be essential both for cytokinesis and for completion of the complex Dictyostelium developmental cycle. To explore the biological role of the carboxyl-terminal portion of the myosin tail, we have created a Dictyostelium cell line bearing a mutation designated my delta C34 in the myosin (mhcA) locus. This cell line produces a truncated myosin protein lacking the 34-kDa carboxyl terminus of the wild-type tail. Southern blots of the mutant cells show that the myosin gene was disrupted by homologous recombination of the transforming plasmid into the myosin locus. Based on in vitro studies of myosin functional domains, the 200-kDa truncated myosin was designed to include a domain important for assembly but to eliminate a domain important for threonine phosphorylation. The mutant cells are defective in cytokinesis, similar to those mutants that are either devoid of myosin (null cells) or contain a truncated 140-kDa myosin (hmm cells). However, unlike previous mutants, the cells carrying the my delta C34 mutation are able to complete the Dictyostelium developmental cycle to form fruiting bodies. Thus a truncated 200-kDa myosin can substitute for native myosin to function in developing cells. These results demonstrate that the 34-kDa carboxyl terminus of myosin, which contributes regulated phosphorylation sites and 20% of the total length of the rod, is not required for the developmental cycle of Dictyostelium.

Expression of Dictyostelium myosin tail segments in Escherichia coli: domains required for assembly and phosphorylation.

The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM-34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.