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PubMed1970

Rapid axonal transport of sulfated mucopolysaccharide proteins.

When sulfur-35-labeled sodium sulfate is injected intraocularly in the goldfish, labeled sulfated mucopolysaccharides rapidly appear in the contra-lateral optic tectum of the brain, demonstrating the axonal flow of sulfated mucopolysaccharides. The transport rate is the same as that observed for proteins labeled after intraocular injection of tritiated proline. Treatment of the sulfur-35-labeled material with precipitants and enzymes reveals the presence of substances with properties similar to those of heparan sulfate (the major component) and chondroitin sulfate. Dermatan sulfate was not detected.

Animals↗
PubMed1973

Use of cobalt as a mitochondrial vital stain to study cytoplasmic exchange in matings of the basidiomycete Schizophyllum commune.

Differential labeling of mates, based on the selective uptake of cobalt by mitochondria of one of the partners, has been used to determine visually by means of phase-contrast microscopy whether transfer of mitochondria occurs after hyphal fusion. Compatible and incompatible matings of the tetrapolar basidiomycete, Schizophyllum commune, were studied. Transfer was detectable in common-A, common-AB, and fully compatible matings. It was not detectable in common-B matings

Basidiomycota↗
PubMed1986

Metabolism and control of lipid structure modification.

The lipid composition characteristic of a particular cellular membrane can become significantly altered, sometimes quite suddenly, when the cell is placed under environmental stress. In the majority of cases examined, the alterations seem to return the membrane's physical state towards that existing prior to imposition of the stress. The compositional changes are often diverse in their nature and also in their site of origin within the cell. Certain modifications, such as changes in the degree of phospholipid acyl chain unsaturation and in the reordering of fatty acid pairing on specific phospholipids, are now recognized as crucial first responses to stress, while others (e.g., fluctuations in relative proportions of different phospholipid classes and in the sterol:phospholipid ratio) develop more slowly and may represent secondary adjustments to the initial lipid changes. The factors directly responsible for modifying membrane lipid composition are generally unknown at the molecular level, but recent advances provide new clues favoring involvement, in some cases, of the ubiquitous mediator Ca2+. In other cases, the physical state of a membrane may directly modulate the activity of lipid-metabolizing enzymes embedded therein.

Cell Membrane↗
PubMed2003

The dynamic and motile properties of intermediate filaments.

For many years, cytoplasmic intermediate filaments (IFs) were considered to be stable cytoskeletal elements contributing primarily to the maintenance of the structural and mechanical integrity of cells. However, recent studies of living cells have revealed that IFs and their precursors possess a remarkably wide array of dynamic and motile properties. These properties are in large part due to interactions with molecular motors such as conventional kinesin, cytoplasmic dynein, and myosin. The association between IFs and motors appears to account for much of the well-documented molecular cross talk between IFs and the other major cytoskeletal elements, microtubules, and actin-containing microfilaments. Furthermore, the associations with molecular motors are also responsible for the high-speed, targeted delivery of nonfilamentous IF protein cargo to specific regions of the cytoplasm where they polymerize into IFs. This review considers the functional implications of the motile properties of IFs and discusses the potential relationships between malfunctions in these motile activities and human diseases.

Animals↗
PubMed2003

Pigment cells: a model for the study of organelle transport.

Eukaryotic organisms rely on intracellular transport to position organelles and other components within their cells. Pigment cells provide an excellent model to study organelle transport as they specialize in the translocation of pigment granules in response to defined chemical signals. Pigment cells of lower vertebrates have traditionally been used as a model for these studies because these cells transport pigment organelles in a highly coordinated fashion, are easily cultured and transfected, are ideal for microsurgery, and are good for biochemical experiments, including in vitro analysis of organelle motility. Many important properties of organelle transport, for example, the requirement of two cytoskeletal filaments (actin and microtubules), the motor proteins involved, and the mechanisms of their regulation and interactions, have been studied using pigment cells of lower vertebrates. Genetic studies of mouse melanocytes allowed the discovery of essential elements involved in organelle transport including the myosin-Va motor and its receptor and adaptor molecules on the organelle surface. Future studies of pigment cells will contribute to our understanding of issues such as the cooperation among multiple motor proteins and the mechanisms of regulation of microtubule motors.

Animals↗
PubMed1991

Astrotactin provides a receptor system for CNS neuronal migration.

CNS neuronal migration is a specialized form of cell motility that sets forth the laminar structure of cortical regions of brain. To define the neuronal receptor systems in glial-guided neuronal migration, an in vitro assay was developed for mouse cerebellar granule neurons, which provides simultaneous tracking of hundreds of migrating neurons. Three general classes of receptor systems were analyzed, the neuron-glial adhesion ligand astrotactin, the neural cell adhesion molecules of the IgG superfamily, N-CAM, L1 and TAG-1, and the beta 1 subunit of the integrin family. In the absence of immune activities, migrating cerebellar granule neurons had an average in vitro migration rate of 12 microns h-1, with individual neurons exhibiting migration rates over a range between 0 to 70 microns h-1. The addition of anti-astrotactin antibodies (or Fabs) significantly reduced the mean rate of neuronal migration by sixty-one percent, resulting in eighty percent of the neurons having migration rates below 8 microns h-1. By contrast, blocking antibodies (or Fabs) against L1, N-CAM, TAG-1 or beta 1 integrin, individually or in combination, did not reduce the rate of neuronal migration. By video-enhanced contrast differential interference contrast microscopy the effects of anti-astrotactin antibodies were seen to be rapid. Within fifteen minutes of antibody application, streaming of cytoplasmic organelles into the leading process arrested, the nucleus shifted from a caudal to a central position, and the extension of filopodia and lamellopodia along the leading process ceased. Correlated video and electron microscopy suggested that the mechanism of arrest by antiastrotactin antibodies involved the failure to form new adhesion sites along the leading process and the disorganization of cytoskeletal components. These results suggest astrotactin acts as a neuronal receptor for granule neuron migration along astroglial fibers.

Animals↗
PubMed1995

Drosophila nonmuscle myosin II is required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos.

The X-linked Drosophila gene spaghetti squash (sqh) encodes the regulatory light chain of nonmuscle myosin II. To assess the requirement for myosin II in oogenesis and early embryogenesis, we induced homozygous germline clones of the hypomorphic mutation sqh1 in otherwise heterozygous mothers. Developing oocytes in such sqh1 germline clones often failed to attain full size due to a defect in 'dumping', the rapid phase of cytoplasmic transport from nurse cells. In contrast to other dumpless mutants described to date, sqh1 egg chambers showed no evidence of ring canal obstruction, and no obvious alteration in the actin network. However the distribution of myosin II was abnormal. We conclude that the molecular motor responsible for cytoplasmic dumping is supplied largely, if not exclusively, by nurse cell myosin II and we suggest that regulation of myosin activity is one means by which cytoplasmic transport may be controlled during oocyte development. The eggs resulting from sqh1 clones, though smaller than normal, began development but exhibited an early defect in axial migration of cleavage nuclei towards the posterior pole of the embryo, in a similar manner to that seen in early cleavage eggs in which the actin cytoskeleton is disrupted. Thus both nurse cell dumping and axial migration require a maternally supplied myosin II.

Actins↗
PubMed2002

The growth of Drosophila bristles and laterals is not restricted to the tip or base.

The highly elongated bristles of Drosophila have proven to be a valuable model system for studying cellular morphogenesis. Extending bristles contain a series of large bundles of actin filaments juxtaposed to the plasma membrane and centrally located microtubules. Models to explain the extension of the bristle have principally focused on the assembly of actin filaments at the distal tip of the bristle. We have used time-lapse observations of wild-type and mutant bristles and the related arista laterals and come to the conclusion that growth takes place throughout the growing cellular extension. This distributed growth can explain the behavior of split laterals and the shape changes seen at the tip during bristle and lateral outgrowth. Inhibitor studies suggest that the microtubule cytoskeleton is essential for maintaining the highly biased axial growth of these structures. We have used fluorescence recovery after photo-bleaching to study the dynamics of the cytoskeleton during bristle growth. Our experiments show that actin bundles in growing bristles are quite stable and move in a retrograde fashion. The bristle microtubules are less stable. The retrograde movement of the peripheral actin appears to be counterbalanced by the distally directed movement of cytoplasm in the center of the bristle.

Actin Cytoskeleton↗
PubMed2003

Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension.

The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal approximately 30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.

Actin Cytoskeleton↗
PubMed2004

Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy.

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of alpha5-integrin in migrating cells. Alpha5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is approximately 1.4 times more concentrated, approximately 4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are approximately 900 integrins microm(-2) (about fivefold higher than surrounding regions). Interestingly, alpha5-integrin and alpha-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, alpha-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 microm min(-1)), while all of the paxillin diffuses away rapidly.

Actinin↗
PubMed2005

Role of the spindle-pole-body protein ApsB and the cortex protein ApsA in microtubule organization and nuclear migration in Aspergillus nidulans.

Nuclear migration and positioning in Aspergillus nidulans depend on microtubules, the microtubule-dependent motor protein dynein, and auxiliary proteins, two of which are ApsA and ApsB. In apsA and apsB mutants nuclei are clustered and show various kinds of nuclear navigation defects, although nuclear migration itself is still possible. We studied the role of several components involved in nuclear migration through in vivo fluorescence microscopy using fluorescent-protein tagging. Because ApsA localizes to the cell cortex and mitotic spindles were immobile in apsA mutants, we suggest that astral microtubule-cortex interactions are necessary for oscillation and movement of mitotic spindles along hyphae, but not for post-mitotic nuclear migration. Mutation of apsA resulted in longer and curved microtubules and displayed synthetic lethality in combination with the conventional kinesin mutation DeltakinA. By contrast, ApsB localized to spindle-pole bodies (the fungal centrosome), to septa and to spots moving rapidly along microtubules. The number of cytoplasmic microtubules was reduced in apsB mutants in comparison to the wild type, indicating that cytoplasmic microtubule nucleation was affected, whereas mitotic spindle formation appeared normal. Mutation of apsB suppressed dynein null mutants, whereas apsA mutation had no effect. We suggest that nuclear positioning defects in the apsA and apsB mutants are due to different effects on microtbule organisation. A model of spindle-pole body led nuclear migration and the roles of dynein and microtubules are discussed.

Aspergillus nidulans↗