A technique for the study of human prostatic epithelial cells in vitro by time-lapse cinematography

The locomotory invasive ability of HeLa cells was tested against: (a) embryonic chick heart fibroblasts (CHF); and (b) normal epithelial cells from human cervix (HCE) in explant confrontations. Data for analyses were obtained from replicate cultures fixed 24 h after junction and from 24-h time-lapse films. The mean invasion index for HeLa versus CHF did not indicate significant obstruction but analyses of hourly radial advance and orientation frequencies showed that obstruction eventually developed as postjunctional incubation time increased. Early contacts between HeLa and CHF demonstrated non-reciprocity of type I contact inhibition of locomotion by the tumour cells, which continued moving in their original direction to underlap contact-inhibited fibroblasts and eventually to occupy spaces vacated by them. When CHF population density increased and free space diminished, HeLa cells displayed directional and probably substrate-dependent contact inhibition. The high invasion index of HeLa versus HCE was largely due to occupation of previous HCE territory by tumour cells and only occasionally to actual infiltration of the epithelial sheet. After contact with HeLa, ruffling substrate-adherent marginal epithelial cells displayed contractile, type I contact inhibition of locomotion. After orientation changes, they gradually retreated. Against HCE, HeLa cells exhibited non-reciprocity of type I contact inhibition and continued radially forward, following the retreating epithelial margin. They did not move onto exposed upper surfaces of epithelial cells and did not underlap marginal cells firmly adherent to the substratum. Invasion of the epithelial sheet was seen only when initial access beneath a cell with a non-adherent margin was available. The contact relationships of isolated invading HeLa cells with their epithelial neighbours suggested successive non-reciprocal contact inhibition reactions.

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How we discovered fluorescent speckle microscopy

Molecular Biology of the Cell ◽

10.1091/mbc.e11-07-0646 ◽

2011 ◽

Vol 22 (21) ◽

pp. 3940-3942 ◽

Cited By ~ 2

Author(s):

E. D. Salmon ◽

Clare M. Waterman

Keyword(s):

Epithelial Cells ◽

Fluorescence Intensity ◽

Focal Adhesions ◽

Time Lapse ◽

Living Cells ◽

Variable Intensity ◽

Macromolecular Assemblies ◽

Dynamic Assembly ◽

Fluorescence Images

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ∼10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual “speckled” appearance—there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

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Regulation of Myosin II Dynamics by Phosphorylation and Dephosphorylation of Its Light Chain in Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0590 ◽

2007 ◽

Vol 18 (2) ◽

pp. 605-616 ◽

Cited By ~ 98

Author(s):

Toshiyuki Watanabe ◽

Hiroshi Hosoya ◽

Shigenobu Yonemura

Keyword(s):

Epithelial Cells ◽

Light Chain ◽

Fluorescent Protein ◽

Myosin Ii ◽

Time Lapse ◽

Stress Fibers ◽

Interacting Protein ◽

Cytoskeleton Organization

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.

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A Technique for the Study of Human Prostatic Epithelial Cells in Vitro by Time-lapse Cinematography

The Journal of Urology ◽

10.1016/s0022-5347(17)61783-x ◽

1970 ◽

Vol 104 (4) ◽

pp. 564-571 ◽

Cited By ~ 5

Author(s):

Virginia L. Lerch ◽

Jean Todd ◽

John K. Lattimer ◽

Myron Tannenbaum

Keyword(s):

Epithelial Cells ◽

Time Lapse

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Non-reciprocal contact inhibition of locomotion of chick embryonic choroid fibroblasts by pigmented retina epithelial cells

Journal of Cell Science ◽

10.1242/jcs.33.1.103 ◽

1978 ◽

Vol 33 (1) ◽

pp. 103-120

Author(s):

E.K. Parkinson ◽

J.G. Edwards

Keyword(s):

Epithelial Cells ◽

Light And Electron Microscopy ◽

Leading Edge ◽

Time Lapse ◽

Contact Inhibition ◽

Cultured Fibroblasts ◽

Lateral Contact ◽

Fibroblastic Cells

Using light and electron microscopy, we have confirmed an earlier observation that chick embryonic pigmented retina epithelial cells (PRE cells) seeded in vitro on cultured sheets of choroid fibroblasts, are able to spread. Spreading is as rapid (and shows the same dependence on lateral contact between PRE cells) as on a serum-coated culture substrate. After 1 h most cells are spreading on the upper surface of the choroid sheet, but after 4 h, some PRE cells can be found sandwiched between overlapping choroid cells, and thus have invaded the sheet. Choroid fibroblasts underlie PRE in vivo, but the ability of PRE cells to spread on cultured fibroblasts is not specific for choroid, since PRE cells spread also on BKH21 hamster kidney fibroblasts, and on fibroblasts from chick embryonic heart. As reported by others for various fibroblastic cells, choroid cells seeded on to choroid sheets or on to cultured PRE are unable to spread. A possible explanation is that spreading of adherent cells is contact-inhibited by the cells in the sheet, just as their leading edges are paralysed on contact, and thus locomotion is inhibited, when fibroblasts collide on a plane substratum. If spreading of seeded cells and cell locomotion are inhibited by the same mechanism, PRE cells should contact-inhibit choroid fibroblasts with which they collide, but not themselves be so inhibited. Using time-lapse cinemicrography, we have found this to be the case. We first established that in homotypic collisions, choroid fibroblasts do show contact inhibition of locomotion, despite the criss-cross (not well monolayered) appearance of confluent cultures. In heterotypic collisions between choroid and PRE we found the predicted nonreciprocal behaviour: the choroid leading edge is paralysed on collision, and the cell subsequently retracts, whereas the active PRE margin appears to be completely unaffected. Speed measurements from a series of such collisions show that the speed of choroid cells is markedly reduced on collision with PRE, whereas the slight slowing of PRE is not statistically significant. We have observed similar behaviour in heterotypic collisions between various epithelial and fibroblastic cells, and so it seems possible that non-reciprocity may prove general for this interaction. If so, it has important implications for the role of contact inhibition of locomotion in phenomena such as morphogenesis, wound healing and the invasiveness of carcinoma cells. On the one hand, non-reciprocal contact-inhibition of locomotion may permit the spreading of epithelia over mesenchymal cells, thus generating or restoring an epithelial bounding membrane. On the other hand, in the absence of other interactions, it would fail to inhibit the invasion of mesenchymal territory by aberrant epithelial cells, or presumably by epithelial free edges.

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

1978 ◽

Vol 36 (2) ◽

pp. 650-651

Author(s):

Raul I. Garcia ◽

Evelyn A. Flynn ◽

George Szabo

Keyword(s):

Direct Injection ◽

Skin Pigmentation ◽

Freeze Fracture ◽

Pigment Granule ◽

Time Lapse ◽

Ultrastructural Level ◽

Lanthanum Tracer ◽

A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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Electron probe x-ray microanalysis and electron microscopy of amino acid absorption and transport by the small intestine: inhibition by chemical poisons and correlation to the elemental composition of the epithelial cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142311 ◽

1986 ◽

Vol 44 ◽

pp. 134-135

Author(s):

A. J. Tousimis

Keyword(s):

Small Intestine ◽

Amino Acid ◽

Epithelial Cells ◽

Elemental Composition ◽

Isotope Labeling ◽

Structural Components ◽

X Ray ◽

Human Small Intestine ◽

Transport Studies

The elemental composition of amino acids is similar to that of the major structural components of the epithelial cells of the small intestine and other tissues. Therefore, their subcellular localization and concentration measurements are not possible by x-ray microanalysis. Radioactive isotope labeling: I131-tyrosine, Se75-methionine and S35-methionine have been successfully employed in numerous absorption and transport studies. The latter two have been utilized both in vitro and vivo, with similar results in the hamster and human small intestine. Non-radioactive Selenomethionine, since its absorption/transport behavior is assumed to be the same as that of Se75- methionine and S75-methionine could serve as a compound tracer for this amino acid.

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