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At least 1,099 records · Page 61Linked to original sources
PubMed2005

Recognition-induced polymersomes: structure and mechanism of formation.

Random polystyrene copolymers grafted with complementary recognition elements were combined in chloroform producing vesicular aggregates, that is, recognition-induced polymersomes (RIPs). Reflection interference contrast microscopy (RICM) in solution, coupled with optical microscopy (OM) and atomic force microscopy (AFM) on solid substrates, were used to determine the wall thickness of the RIPs. Rather than a conventional mono- or bilayer structure (approximately 10 or approximately 20 nm, respectively) the RIP membrane was 43+/-7 nm thick. Structural arrangement of the polymer chains on the RIP wall were characterized by using angle-resolved X-ray photoelectron spectroscopy (AR-XPS). The interior portion of the vesicle membrane was found to be more polar, containing more recognition units, than the exterior part. This gradient suggests that a rapid self-sorting of polymers takes place during the formation of RIPs, providing the likely mechanism for vesicle self-assembly.

Journal Article↗
PubMed2004

Curcumin inhibits cell motility and alters microfilament organization and function in prostate cancer cells.

Curcumin is a dietary phytochemical associated with anti-tumorigenic effects, but the mechanisms by which it inhibits cancer cell growth and metastasis are not completely understood. For example, little information is available regarding the effects of curcumin on cytoskeletal organization and function. In this study, time-lapse video and immunofluorescence labeling methods were used to demonstrate that curcumin significantly alters microfilament organization and cell motility in PC-3 and LNCaP human prostate cancer cells in vitro. Curcumin rapidly arrests cell movements and subsequently alters cell shape in the highly motile PC-3 cell line, but has a less noticeable effect on the relatively immobile LNCaP cell line. Stress fibers are augmented, and the overall quantity of f-actin appears to increase in both types of cells following curcumin treatment. Cytochalasin B (CB) disrupts microfilament organization in both cell lines, and causes vigorous membrane blebbing in PC-3 cells, but not LNCaP cells. Pre-treatment of cells with curcumin suppresses changes in microfilament organization caused by CB, and blocks PC-3 membrane blebbing. At least some of the effects of curcumin appear to be mediated by protein kinase C (PKC), as treatment with the PKC inhibitor bisindolylmaleimide inhibits the ability of curcumin to block CB-induced membrane blebbing. These findings demonstrate that curcumin exerts significant effects on the actin cytoskeleton in prostate cancer cells, including altering microfilament organization and function. This is a novel observation that may represent an important mechanism by which curcumin functions as a chemopreventative agent, and as an inhibitor of angiogenesis and metastasis.

Actin Cytoskeleton↗
PubMed1981

Video-enhanced contrast polarization (AVEC-POL) microscopy: a new method applied to the detection of birefringence in the motile reticulopodial network of Allogromia laticollaris.

A new method is described for recording rapid processes of cell motility in polarized light. The Allen video-enhanced contrast (AVEC-POL) method of polarization microscopy achieves significant improvements in resolution, contrast, and the visibility of fine detail by a combination of novel adjustments to a standard (unrectified) polarizing microscope and video camera. Using the full working aperture of a high-power planapochromatic objective lens and compensator setting of lambda/9-lambda/4, visible images appear lacking in contrast. However, the same images viewed with an appropriate video camera equipped with an electronic offset adjustment can be made to appear with as much contrast as desired, revealing a significantly greater amount of fine detail in the image than can be seen by high extinction visual microscopy alone. At bias retardations between one-ninth and one-quarter wave, the diffraction anomaly observed near extinction disappears. Consequently, polarizing rectifiers are not required with the AVEC-POL method, and images previously requiring photographic exposures of around 20 seconds are sufficiently bright to be registered on the video monitor in 1/60 second. Using an intensity monitor, quantitative measurements of cellular birefringence can be retrieved from live or videotaped images displaying a linear relationship between contrast and phase retardation due to birefringence. The AVEC-POL method also renders accessible to polarized light analysis a number of objects that scatter or depolarize too much light to be studied by high extinction methods. The method is demonstrated on model objects and applied to the highly motile reticulopodial network of Allogromia laticollaris. Rapid motion in close association with microtubules can now be analyzed in greater detail at a significant reduction in the cost of recording.

Animals↗
PubMed1984

Direct visualization of particle velocity distribution by pseudostereoscopic viewing of time-lapsed sequential images: application to fast axonal transport.

We describe a simple method for direct visualization of the velocity distribution of particles moving against an immobile background. The technique involves pseudostereoscopic viewing of image pairs separated by an appropriate time interval in a sequential recording of the subject. Under these conditions, the positive or negative parallax arising from particle motion results in the binocular image of a particle being perceived as raised or lowered relative to an immobile background plane depending on its direction of movement, and with the degree of perceived elevation being proportional to its speed. In effect, the binocular optic axis becomes a velocity (speed) axis under these conditions. The technique is illustrated with examples of image pair sequences showing fast axonal transport in lobster and squid axons using video-enhanced differential interference contrast microscopy. However, the pseudostereoscopic method is quite generally applicable to both microscopic and macroscopic time-dependent phenomena. Particle speeds can be quantitated using standard procedures for measuring frame-to-frame particle displacements, or alternatively, by determination of parallax using stereogrammatic methods. It should be also readily adaptable for on-line monitoring of particle velocity distribution, particularly in video systems where frame buffers can be utilized to extract and present serial image pairs having any desired time separation from video-taped sequences.

Animals↗
PubMed1986

Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles.

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.

Acrylamide↗
PubMed1986

Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos.

In this report, we describe an in vitro system for analyzing microtubule-based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol-stabilized microtubules assembled in low speed supernatants of Lytechinus egg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (-) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (-) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin-like. The implications of these molecular interactions for mitosis and other motile events are discussed.

Alkaloids↗
PubMed1988

Micromanipulation studies of the asymmetric positioning of the maturation spindle in Chaetopterus sp. oocytes: I. Anchorage of the spindle to the cortex and migration of a displaced spindle.

We investigated the nature of the asymmetric positioning and attachment of Chaetopterus oocyte meiotic spindles to the animal pole cortex by micromanipulation. The manipulated spindle's behavior was analyzed in clarified oocyte fragments using video-enhanced polarized light microscopy. As the spindle was drawn towards the cell interior with a microneedle, the cell surface dimpled inwards adjacent to the outer spindle pole. As the spindle was pulled further inwards, the dimple suddenly receded indicating a rupture of a mechanical link between the cell cortex and outer spindle pole. The spindle paused briefly when released from the microneedle; then it spontaneously migrated back to the original attachment site and reassociated with the cell cortex. Positive birefringent astral fibers were seen running between the outer spindle pole and the cortex during the migration. The velocity of the spindle during its migration tended to increase as it came closer to the cortex. Velocities as high as 1.25 micron/sec. were measured. If removed too far from the attachment site cortex (greater than 35 micron), the spindle remained stationary until pushed closer to the original attachment site. Spindles, inverted by micromanipulation, migrated and reattached to the cortical site by their former inner pole; thus either spindle pole can seek out and migrate to the original attachment site. However, spindle poles pushed against other cortical regions did not attach demonstrating that there is only one unique, localized attachment site for spindle attachment.

Animals↗
PubMed1990

Formation, transport, contraction, and disassembly of stress fibers in fibroblasts.

Swiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum-deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video-enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive manner at the cytoplasm-membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10-20 microns/h. When serum-deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post-stimulation.

Actins↗
PubMed1991

Dynamic morphology of metastatic mouse T-lymphoma cells invading through monolayers of 10T1/2 cells.

We have used an in vitro model system to analyze cytomechanical aspects of tissue infiltration by T-lymphocytes. The interaction of metastatic T-lymphoma cells with a precultured monolayer of 10T1/2 fibroblast-like cells was recorded in time-lapse video with alternating phase contrast and reflection interference contrast microscopy. Sectioning of embedded specimens as well as cytoskeletal stainings have been performed on matching cocultures. The lymphoma cells did not strongly attach or spread on the dorsal surface of the monolayer cells. Invasion started with the protrusion of a pseudopodium through a narrow gap, and conspicuous constriction of the invading cell's body and nucleus was a consistent feature during the later steps. Overt retraction of the target cells was not seen, but the invading lymphoma cells elevated the fibroblasts over relatively large areas, thereby creating dome-shaped open spaces, allowing for further migration under the monolayer with minimal resistance. Invasion was not unidirectional but was readily reversible at any stage. Due to this wavering character, an invasion event could take more than 1 hour, although the shape alterations involved were fast. Even after the invasion process had been completed, the lymphoma cells could come out from below the monolayer again. Therefore we propose that invasion in this model should be considered as a dynamic equilibrium. Invading T-lymphoma cells displayed diffuse F-actin staining and a well-organized microtubular complex with the centrosomes behind the nucleus in the uropod, which also contained most vesicular organelles.

Actins↗
PubMed1992

End-stabilized microtubules observed in vitro: stability, subunit, interchange, and breakage.

We report a reliable method to prepare, in vitro, microtubules that are stabilized at both ends by axonemal structures, and report studies of their properties. Such "end-stabilized" microtubules neither grow nor shorten over times of several hours when tubulin subunits are present in the surrounding solution. When subunits are removed, the microtubules eventually break. Breakage occurs within a sinuous and flexible region, a few microns in length, that begins at a single point on the microtubule and grows. When breakage does occur, the resulting two free ends shorten very rapidly until the flexible part has depolymerized and the region of straight microtubule is reached. The remainder of the microtubule then shortens at rates comparable to those ordinarily observed in dynamic instability. Formation of the flexible region can be reversed if subunits are added to the buffer prior to breakage. End-stabilized microtubules are a useful tool for studying interactions of molecules with the microtubular wall. They may be a good model for interpreting stabilizing events that happen in the cell. A preliminary study of the effects of microtubule poisons on the wall is presented.

Alkaloids↗
PubMed1993

Motility of intracellular particles in rat fibroblasts is greatly enhanced by phorbol ester and by over-expression of normal p21N-ras.

Particle motility in cultured rat fibroblasts was studied using video-enhanced differential interference contrast microscopy. The average velocity of large bright particles (apparent diameter about 0.5-0.7 micron) was measured in control cells and in cells treated with agents which affected targets related to signal transduction pathways. A Rat-2-derived fibroblast line transfected with a construct containing multiple copies of the N-ras proto-oncogene under the control of dexamethasone-sensitive promoter was used as a main experimental model. Dexamethasone treatment was shown to induce high levels of N-ras expression in these cells. This treatment greatly increased the average particle velocity. At the same time dexamethasone did not influence the particle motility in the non-transfected parent cells and in the cells transfected with a construct which did not contain N-ras. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), also induced an approximate eightfold increase in the particle rate after several hours of incubation, while sphingosine, an inhibitor of PKC, prevented this activation. Sphingosine alone reduced the particle motility after a 20 min incubation. The particle movements were inhibited also by colcemid. These data show that the activation of N-ras and PKC produced dramatic activation of microtubule-dependent particle motility. A possible role of this activation in signal-induced alterations of cell morphology is discussed.

Animals↗
PubMed1993

Unambiguous classification of microtubule-ends in vitro: dynamic properties of the plus- and minus-ends.

To understand the mechanism of dynamic instability of microtubule growth and shortening, one needs a means of reliably determining the polarity of the microtubules under investigation. Sea urchin sperm-tail axonemal fragments nucleate the growth of both plus-ended and minus-ended microtubules, but their polarity is not apparent by video-enhanced DIC microscopy. The polarity of a microtubule is usually assessed by observing differences between the rates and lengths of growth and shortening excursions of the two ends. In practice, though, a significant fraction of the population of microtubules displays characteristics intermediate between the average characteristics of either end, thereby escaping classification. Excluding these "intermediate" microtubules from the measured populations introduces bias into the understanding of microtubule dynamic instability. We circumvent this problem by making use of the plus-end directed movement of the microtubule-dependent molecular motor kinesin to determine the polarity of any given microtubule unambiguously. Carboxylated-microspheres coated with kinesin, which are clearly visible by DIC microscopy, were used to determine the polarity of a microtubule. The dynamics were then observed. Kinesin was found to have no marked effect on dynamic instability. By this technique, we show that the distributions of properties that describe microtubule dynamic instability (rates and lengths of growth and shortening as well as frequencies of interconversion between these phases) of plus-ends overlap to a significant extent with those of minus-ends. It is this overlap that obscures the usual classification of the ends. Therefore, models describing microtubule dynamic instability need to incorporate the broad and overlapping range of properties of the two ends.

Animals↗
PubMed1994

Microtubules with altered assembly kinetics have a decreased rate of kinesin-based transport.

Microtubules treated with the gamma-diketone 2,5-hexanedione (2,5-HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5-HD-induced microtubule modification. 2,5-HD-treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin-based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5-HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3-Acetyl-2,5-hexanedione (AcHD), a pyrrole forming, non-crosslinking congener of 2,5-HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5-HD. However, glutaraldehyde, a pyrrole-independent crosslinking agent which alters microtubule assembly in the same way as 2,5-HD, slowed microtubule transport. These results indicate that a 2,5-HD-induced microtubule modification, possibly a crosslink-related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule-motor interaction.

Animals↗
PubMed1995

beta-Tubulin mutation suppresses microtubule dynamics in vitro and slows mitosis in vivo.

Microtubule (MT) dynamics vary both spatially and temporally within cells and are thought to be important for proper MT cellular function. Because MT dynamics appear to be closely tied to the guanosine triphosphatase (GTPase) activity of beta-tubulin subunits, we examined the importance of MT dynamics in the budding yeast S. cerevisiae by introducing a T107K point mutation into a region of the single beta-tubulin gene, TUB2, known to affect the assembly-dependent GTPase activity of MTs in vitro. Analysis of MT dynamic behavior by video-enhanced differential interference contrast microscopy, revealed that T107K subunits slowed both the growth rates and catastrophic disassembly rates of individual MTs in vitro. In haploid cells tub2-T107K is lethal; but in tub2-T107K/tub2-590 heterozygotes the mutation is viable, dominant, and slows cell-cycle progression through mitosis, without causing wholesale disruption of cellular MTs. The correlation between the slower growing and shortening rates of MTs in vitro, and the slower mitosis in vivo suggests that MT dynamics are important in budding yeast and may regulate the rate of nuclear movement and segregation. The slower mitosis in mutant cells did not result in premature cytokinesis and cell death, further suggesting that cell-cycle control mechanisms "sense" the mitotic slowdown, possibly by monitoring MT dynamics directly.

Base Sequence↗
PubMed2003

Coordinated development of identified serotonergic neurons and their target ciliary cells in Helisoma trivolvis embryos.

Embryonic neuron C1s (ENC1s) are bilateral serotonergic neurons that function as cilioexcitatory motor neurons in embryonic development of the pond snail, Helisoma trivolvis. Recent experiments demonstrated that these neurons stimulate cilia-driven embryo rotation in response to hypoxia. In the present study, a comprehensive anatomic analysis of these cells and their target ciliary structures was done to address the following questions: (1) Does ENC1 have a morphology consistent with an oxygen-sensitive sensory cell; (2) Is the development of ENC1's neurite outgrowth pathway coordinated with the development of its target effectors, the pedal and dorsolateral ciliary bands; and (3) What is the anatomic basis of ENC1-ciliary communication? By using an array of microscopic techniques on live and serotonin-immunostained embryos, we found that each ENC1 possessed an apical dendrite that was capped with an integral dendritic knob penetrating the embryo surface. The dendritic knobs contained both microvilli and nonmotile cilia that suggested a sensory transduction role. Each ENC1 also possessed a descending projection, whose development was characterized by the rapid formation of the primary neurite pathway between stages E13 and E15, with the primary neurite of the right ENC1 developing in advance of its contralateral homologue. Secondary neurite branches formed between stages E15 and E30 in a spatiotemporal pattern that closely matched the development of the dorsolateral and pedal bands of cilia. Both dorsolateral and pedal ciliated cells formed basal processes that contacted ENC1 neurites. Finally, gap junction profiles were observed at neurite-neurite, neurite-ciliary cell, and ciliary cell-ciliary cell apposition sites, whereas putative chemical synaptic profiles were observed at neurite-neurite and neurite-ciliary cell apposition sites.

Afferent Pathways↗
PubMed1978

Myelin sheath thickness and conduction latency groups in the cat optic nerve.

In many animals, the optic nerve has multiple conduction latency groups of fibres yet displays a unimodal frequency distribution of fibre sizes. An attempt has been made to resolve this discrepancy in the cat. Examination of teased preparations of fixed cat optic nerve fibres by polarizing interference light microscopy indicated that a fibre's size may change abruptly along its length. Furthermore, the diameters of single nerve fibres followed for distances of up to 30 mum in serial transverse sections of cat optic nerve showed variations of up to 100% of a fibre's average diameter over that distance. On the other hand, the myelin sheath thickness of these fibres appeared to be relatively constant along any one fibre. A population of cat optic nerve fibres which had a unimodal axon diameter frequency distribution was found to have a myelin sheath thickness frequency distribution with five modes. These modes have been interpreted as indicating the existence of five groups of fibres in the cat optic nerve. Four of these groups may be related to four negative peaks seen in the antidromic compound action potential recorded at the margin of the cat optic disc following stimulation of the optic chiasm. The fifth myelin thickness group appears to represent the slowly conducting fibres which do not make an obvious contribution to the antidromic compound action potential. It is concluded that for the cat optic nerve, the conduction latency groups seen in the compound action potential may be more directly correlated with the frequency distribution of fixed nerve myelin thickness than with the frequency distribution of fixed nerve fibre diameter.

Action Potentials↗
PubMed1995

Organic material concentration in auditory outer hair cells measured by laser interferometry.

Outer hair cells (OHC) of the mammalian cochlea are quasicylindrical cells of different length, which play a major role in hearing at threshold. Their particular shape allows the use of a noninvasive laser interferometric technique of isolated cells in vitro in order to measure the organic material concentration (OMC), hence the density of each cell body. In most (95%) of the OHCs isolated from the same guinea pig, when the cell diameter is normalized, the results show that the cell body OMC does not vary with cell length. In different animals, the respective normalized OMC mean values can vary between 70 kg/m3 and 103 kg/m3. A few OHCs with morphological particularities often possess cell body OMCs > 103 kg/m3. The results of the interferometric measurements in isolated OHCs confirm that density variations in the cell bodies are not involved in a sound frequency coding. The in vitro OMC variations of the OHCs could be related to the isolation procedure; however, they could also correlate with actual in vivo OMC variations.

Animals↗
PubMed2006

In vivo cytometry: a spectrum of possibilities.

BACKGROUND: We investigate whether optical imaging can reliably detect abnormalities in tissue, in a range of specimens (live cells in vitro; fixed, fresh ex-vivo and in vivo tissue), without the use of added contrast agents, and review our promising spectral methods for achieving quantitative, real-time, high resolution intrasurgical optical diagnostics. METHODS: We use reflectance, fluorescence, two-photon, and Mie scattering imaging, performed with instrumentation we developed or modified, to detect intrinsic tissue signatures. Emphasis is on spectral/hyperspectral imaging approaches allowing the equivalent of in vivo pathology. RESULTS: With experimental focus on unstained specimens, we demonstrate the ability to segment tissue images for cancer detection. Spectral reflectance imaging, coupled with advanced analysis, typically yields 90% specificity and sensitivity. Autofluorescence is also shown to be diagnostically useful, with lymph nodes results highlighted here. Elastic scattering hyperspectral imaging endoscopy, using a new instrument we designed and built, shows promise in bronchoscopic detection of dysplasia and early cancer in patients. CONCLUSIONS: The results demonstrate that advanced optical imaging can detect and localize cellular signatures of cancer in real-time, in vivo, without the use of contrast agents, in animals and humans. This is an important step towards tight spatio-temporal coupling between such detection and clinical intervention.

Animals↗