Biomedical subjects

C Aguado-Velasco

Publications and source records attributed to C Aguado-Velasco.

8 recordsLinked to original sources

PubMed1999

Circulation of the plasma membrane in Dictyostelium.

We have developed a fluorimetric assay with the use of the dye FM1-43 to determine the rate at which Dictyostelium amoebae endocytose their surface membrane. Our results show that they do so about once each 4-10 min. A clathrin null mutant takes its surface up only approximately 30% more slowly, showing that this membrane uptake cannot be caused by clathrin-coated vesicles. Surprisingly, Ax2 and its parent, NC4, which differ in their rates of fluid-phase internalization by approximately 60-fold, take up their surfaces at the same rates. These results show that, in axenic cells, the uptake of fluid and of surface area are separate processes. The large activity of this new endocytic cycle in both Ax2 and NC4 amoebae appears capable of delivering sufficient new surface area to advance the cells' fronts during migration.

Animals↗

PubMed1998

EGF induces recycling membrane to form ruffles.

KB cells are know to respond to epidermal growth factor (EGF) by producing prodigious ruffles in the plasma membrane within minutes. The signal transduction pathway underlying this effect in fibroblasts is mediated through Rac, a member of the Ras-like family of GTPases. As ruffles are rich in components of the cytoskeleton--particularly in actin and ezrin--it has been suggested that ruffles arise when activated Rac modulates the actin cytoskeleton to push out a membrane protrusion. We set out to see whether the surface of new ruffles arises from neighbouring membrane, or whether it comes from an intracellular pool of endocytosed membrane. If it arose by exocytosis from endosomes, it would be expected to be enriched in those recycling receptors that are concentrated in coated pits in the endocytic side of the cycle. On the other hand, if it arose passively from the adjacent plasma membrane, a uniform distribution of these receptors would be expected. Here, we show that as soon as ruffles appear on KB cells in response to EGF, their membrane surfaces are enriched in both transferrin and low density lipoprotein (LDL) receptors. Both these proteins are known to be selectively concentrated into endosomal membranes by clathrin-mediated endocytosis. Our results reveal that the surfaces of ruffles arise by exocytosis of internal membrane from the endocytic cycle and, therefore, that a primary action of Rac is to redirect the exocytosis of recycling membrane into just those specific sites where ruffles form.

Actins↗

PubMed1998

Membrane traffic during cell locomotion.

The key role played by a polarised endocytic cycle in extending the front of a eukaryotic cell is now becoming established. Highlights that have occurred during the past year include the visualisation of vesicles fusing with the advancing edge of Physarum, directly leading to extensions of the cell; furthermore, the fusion of vesicles at the leading edge in plant pollen tubes appears to be controlled by a small GTPase, Rop 1, which is a plant homologue of the mammalian Rac. In animal cells, Rac appears to help determine where exocytosis occurs on the surface of a polarised cell. These and other observations illuminate how the endocytic cycle assists the locomotory process in animal cells.

Animals↗

PubMed1997

Dictyostelium myosin II null mutant can still cap Con A receptors.

Cross-linked antigens on the surface of a motile cell cap at the trailing end of the cell. In Dictyostelium discoideum, myosin II null mutants have previously been reported to be unable to cap Con A receptors, although they are able to locomote. This finding implicated myosin II as an essential component of the capping mechanism, although not of the machinery for locomotion. Here we show that myosin II null mutants do cap Con A receptors, albeit less efficiently than does wild type. This shows that cap formation is not absolutely dependent on myosin II and that a close mechanistic relationship between capping, particle movement, and cell migration may still exist.

Animals↗

PubMed1996

NDP kinase can modulate contraction of Dictyostelium cytoskeletons.

Extraction of Dictyostelium amoebae with Triton X-100 produces robust cytoskeletons composed mainly of actin and myosin II. These cytoskeletons rapidly contract when mixed with Mg-ATP in simple buffers. The Triton-soluble fraction was found to contain a GTP-dependent activity that prevented contraction by Mg-ATP. This activity was purified, and identified, as nucleoside diphosphate kinase (NDP kinase). The apparent inhibition resulted from pre-contraction of the cytoskeletons. Tightly bound cytoskeletal ADP was presumably phosphorylated, and the resulting ATP powered contraction. NDP kinase appeared to be unique in this capacity, since other regenerating systems did not cause pre-contraction. Reconstitution experiments demonstrated that the kinase must be in physical contact with the cytoskeleton. These results suggest that Dictyostelium NDP kinase is able to channel ATP to the myosin molecule, and this could play a role in directly regulating cytoskeletal contraction or in facilitating contraction under conditions where intracellular ATP concentrations are low. This ability to modulate cytoskeletal contraction could help to explain observations in other systems whereby defects in NDP kinase result in abnormal development or changes in the metastatic potential of cancer cells.

Animals↗

PubMed1995

Arachidonic acid activation of a new family of K+ channels in cultured rat neuronal cells.

1. The presence and properties of K+ channels activated by arachidonic acid were studied in neuronal cells cultured from the mesencephalic and hypothalamic areas of rat brain. 2. Arachidonic acid produced a concentration-dependent (5-50 microM) and reversible activation of whole-cell currents. 3. In excised membrane patches, arachidonic acid applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels whose current-voltage relationships were slightly outwardly rectifying, inwardly rectifying and linear, and whose single channel slope conductances at +60 mV were 143, 45 and 52 pS, respectively. 4. All three currents were K+ selective and blocked by 2 mM Ba2+ but not by other K+ channel blockers such as tetraethylammonium chloride, 4-aminopyridine and quinidine. The outwardly and inwardly rectifying currents were slightly voltage dependent with higher channel activity at more depolarized potentials. 5. Arachidonic acid activated the K+ channels in cells treated with cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and nordihydroguaiaretic acid), indicating that arachidonic acid itself can directly activate the channels. Alcohol and methyl ester derivatives of arachidonic acid failed to activate the K+ channels, indicating that the charged carboxyl group is important for activation. 6. Certain unsaturated fatty acids (linoleic, linolenic and docosahexaenoic acids), but not saturated fatty acids (myristic, palmitic, stearic acids), also reversibly activated all three types of K+ channel. 7. All three K+ channels were activated by pressure applied to the membrane (i.e. channels were stretch sensitive) with a half-maximal pressure of approximately 18 mmHg. The K+ channels were not blocked by 100 microM GdCl3. 8. A decrease in intracellular pH (over the range 5.6-7.2) caused a reversible, pH-dependent increase in channel activity whether the channel was initially activated by arachidonic acid or stretch. 9. Glutamate, a neurotransmitter reported to generate arachidonic acid in striatal neurons, did not cause activation of the K+ channels when applied extracellularly in cell-attached patches. 10. It is suggested that the K+ channels described here belong to a distinct family of ion channels that are activated by either fatty acids or membrane stretch. Although the physiological roles of these K+ channels are not yet known, they may be involved in cellular processes such as cell volume regulation and ischaemia-induced elevation of K+ loss.

Animals↗

PubMed1993

Contraction of reconstituted Dictyostelium cytoskeletons: an apparent role for higher order associations among myosin filaments.

A large number of cellular functions require assembly of actin and myosin and coordinated interactions between the resulting filaments. To better understand the structure and function of one such contractile assembly, we have begun fractionation and reconstitution studies of Dictyostelium cytoskeletons. Isolated cytoskeletons rapidly contracted when mixed with Mg-ATP, and myosin II was essential for this since myosin-depleted (stripped) cytoskeletons failed to contract. Dictyostelium, Acanthamoeba, or skeletal muscle myosins bound to stripped cytoskeletons with equal efficiency, and the Mg-ATPase of all three myosins was stimulated by the cytoskeleton-associated actin. Near neutral pH, however, only the homologous system reconstituted with Dictyostelium myosin contracted, despite the fact that under the same conditions all three myosins bound to myosin-depleted (ghost) muscle myofibrils and restored contractility. Individual Dictyostelium myosin thick filaments have a strong tendency to aggregate and associate end-to-end, and this may be important for functional contraction of cytoskeletons. This suggestion is supported by the observation that under conditions where individual Acanthamoeba myosin filaments aggregated, reconstituted cytoskeletons contracted. None of the solution conditions tested caused rabbit muscle myosin filaments to aggregate or to contract cytoskeletons. Thus higher order associations among individual myosin filaments may be essential for some types of cell motility.

Acanthamoeba↗

PubMed1993

Isolation of myosin from Dictyostelium cytoskeletons.

Dictyostelium has several isoforms of "unconventional" single-headed myosins that do not assemble into filaments (myosin I). In contrast, there is only one form of conventional myosin (myosin II) that self-assembles into bipolar thick filaments, and molecular genetic studies have shown that this myosin is an essential motor protein for several cell functions, including cytokinesis. Myosin II is phosphorylated on both the heavy chain and light chain, and these modifications regulate assembly and ATPase activity. A large fraction of the myosin II remains associated with structures that are resistant to extraction with nonionic detergents (the Triton cytoskeleton). These cytoskeletons retain the ability to contract in an ATP-dependent manner and can easily be isolated by low-speed centrifugation. A method for the rapid purification of myosin II from cytoskeletons is described. This method is simple and avoids the need for sonication, ultracentrifugation of large volumes, and overnight dialysis. The subunit composition, assembly properties, and enzyme activity are similar to those reported for myosin prepared from soluble extracts.

Animals↗