Formation of hyaluronan- and versican-rich pericellular matrix by prostate cancer cells promotes cell motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.     

Slow pulsatile movements of Schwann cells in vitro: a time-lapse cinemicrographic study

Slow pulsatile movements of Schwann cells in vitro were studied quantitatively by using time-lapse cinemicrography. Schwann cells from peripheral nerves of 3-day-old rats were cultured in serum-free medium. Most Schwann cells showed intermittent episodes of pulsatile movement; each episode consisted of one or several contractile pulses. About half of the episodes consisted of a single pulse, and episodes with more than four pulses were rare. The average episode of activity lasted 2.6 min, while the average duration of a single pulse was 1.5 min. The mean quiescent interval between episodes of activity was 3.7 min. Some cells showed no pulsatile activity. Active cells averaged 6.6 episodes/h. The fraction of time which a Schwann cell spent in pulsatile activity varied widely, with an average of 28%. Behavior of Schwann cells in HEPES-buffered Hanks saline was generally similar to that in the complete medium. Raising K+ to 40 mM or Ca++ to 10 mM did not markedly affect the time course of the pulsatile motility, although the contractions were more vigorous in the high Ca++. Pulsatile movement was reversibly inhibited by cytochalasin B and appeared to be potentiated by drugs that disrupt microtubules.     

The morphology of mesangial cells cultured at high density and in collagen gels

Primary mesangial cells (rat) from monolayer cultures of the 6th to 12th passage and permanent SV40 Mes13 cells were grown at high density in organoid culture at the medium/air interphase. After adaptation to the in vitro conditions, both mesangial cell types developed after 7 days a synthesis apparatus (endoplasmic reticulum, Golgi apparatus) and produced matrix which consisted of Lamina densa-like material, collagenous fibrils and filaments. Unspecific contacts, gap junctions and adhesion belts could be demonstrated in the contact areas. Additionally, some cells exhibited thick bundles of actin filaments. A close resemblance of the mesangial cells in high density culture to those in vivo can, therefore, be stated. Hence, they differentiated with regard to their matrix formation, contraction and contact behaviour and can therefore be used for experimental studies within a short culture period of 7 days. Cell aggregates in monolayer culture and in cultures in collagen gels had not differentiated at this stage.     

Factor VIII-related antigen : A pericellular matrix component of cultured human endothelial cells

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of endothelial cells as well.In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with M_r 220–240; 180; 160; 80 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.     

Novel substratum-dependent dendritic morphology and motility in epithelial cells

NADPase activity has been localized in the exocrine pancreas of rat, by cytochemistry according to the procedure of Smith as modified by Clermont et al. With NADP or NADPH as substrate, an intense reaction was detected in one or two intermediary saccules of the Golgi stack. Reaction product was also present in lysosomes, dense bodies and the gland lumen. It was absent from condensing vacuoles and zymogen granules. A very intense reaction was found over a "snake-like" structure not previously reported. These are elongated tubules located in basal and central portions of the acinar cell where they are frequently seen close to the Golgi stack or the basolateral cell surface.     

High-throughput RNAi screening by time-lapse imaging of live human cells

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.     

Individual cell motility studied by time-lapse video recording: influence of experimental conditions

BACKGROUND: Eukaryotic cell motility plays a key role during development, wound healing, and tumour invasion. Computer-assisted image analysis now makes it a realistic task to quantify individual cell motility of a large number of cells. However, the influence of culture conditions before and during measurements has not been investigated systematically. METHODS: We have evaluated intraassay and interassay variations in determinations of cellular speed of fibroblastoid L929 cells and investigated the effects of a series of physical and biological parameters on the motile behavior of this cell line. Cellular morphology and organization of filamentous actin were assessed by means of phase-contrast and confocal laser scanning microscopy and compared to the corresponding motility data. RESULTS: Cell dissociation procedure, seeding density, time of cultivation, and substrate concentration were shown to affect cellular speed significantly. pH and temperature of the medium most profoundly influenced cell motility and morphology. Thus, the mean cell speed was 40% lower at pH 7.25 than at pH 7.6; at 29 degrees C, it was approximately four times lower than at 39 degrees C. CONCLUSION: Of the parameters evaluated, cell motility was most strongly affected by changes in pH and temperature. In general, changes in cell speed were accompanied by alterations in cell morphology and organization of filamentous actin, although no consistent phenotypic characteristics could be demonstrated for cells exhibiting high cell speed.     

Loss of pericellular fibronectin matrix from heterokaryons of normal human fibroblasts and epithelial cells

The expression of fibronectin in heterokaryons of normal human fibroblasts and normal or malignant epithelial cells was studied by indirect immunofluorescence microscopy. Fibroblasts and their homokaryons showed a characteristic pericellular fibronectin matrix, whereas both normal (MDCK) and malignant (HeLa) epithelial cells, and their homokaryons, lacked such a matrix. The fibroblast homokaryons also showed a typical strong, perinuclear cytoplasmic, fibronectin-specific fluorescence. This was much weaker or absent in the MDCK and HeLa cells and their homokaryons. When human fibroblasts were fused with either normal or malignant epithelial cells, no pericellular matrix-like, fibronectin-specific fluorescence could be seen in the heterokaryons. Interestingly, however, a distinct intracellular fluorescence was seen in the heterokaryons, indicating continued production of fibronectin. The results of the present study indicate that both malignant and normal epithelial cells, which do not deposit fibronectin matrix, can cause its loss in heterokaryons with fibroblasts. Thus, discontinued fibronectin matrix formation does not point exclusively to malignancy, but may also reflect the state of differentiation of the parental cells.     

Gap junctions in human synovial cells and tissue

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.     

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

Genetic basis of human female pelvic morphology: a twin study

To examine the relative role of genetic and environmental factors on pelvic morphology, data on 60 pairs of female twins (30 monozygotic (MZ) and 30 dizygotic (DZ)) were analyzed. Fourteen pelvic measurements were normally distributed, and two were not. Association of twin type with the mean value of a trait was found in only 1 out of 8 traits. Heterogeneity of variance between zygosities was observed in 4 pelvic traits (50%), invalidating within-pair estimates of genetic variance for these traits. Evidence of stronger environmental covariance for MZ than DZ twins was observed for only one trait (sitting height iliocristale). A significant genetic component of variation was observed for age at menarche and in the pelvic area. In instances where inequality of variances between zygosities was demonstrated, total among-pair and within-pair mean squares were larger for dizygotic than for monozygotic twins. This is interpreted as evidence of greater environmental influence between zygosities. Environmental modification was not of the same magnitude in various pelvic traits. Bitrochanteric breadth had the highest magnitude of cultural heritability, indicating that cultural factors played an important role in determining hip breadth.     

The dynamic structure of the pericellular matrix on living cells

Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b- HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan- aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.     

Development and morphology of rat synovial membrane

The development of the knee joint and synovial membrane in Wistar rats was studied from the 13th fetal day until adulthood by means of light microscopy, transmission electron microscopy and scanning electron microscopy. Using acid phosphatase and peroxidase methods, the different cell types of synovial membrane could be identified.     

The integrin-linked kinase regulates cell morphology and motility in a rho-associated kinase-dependent manner

The integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transmission from integrin and growth factor receptors. We have determined that ILK regulates U2OS osteosarcoma cell spreading and motility in a manner requiring both kinase activity and localization. Overexpression of wild-type (WT) ILK resulted in suppression of cell spreading, polarization, and motility to fibronectin. Cell lines overexpressing kinase-dead (S343A) or paxillin binding site mutant ILK proteins display inhibited haptotaxis to fibronectin. Conversely, spreading and motility was potentiated in cells expressing the "dominant negative," non-targeting, kinase-deficient E359K ILK protein. Suppression of cell spreading and motility of WT ILK U2OS cells could be rescued by treatment with the Rho-associated kinase (ROCK) inhibitor Y-27632 or introduction of dominant negative ROCK or RhoA, suggesting these cells have increased RhoA signaling. Activation of focal adhesion kinase (FAK), a negative regulator of RhoA, was reduced in WT ILK cells, whereas overexpression of FAK rescued the observed defects in spreading and cell polarity. Thus, ILK-dependent effects on ROCK and/or RhoA signaling may be mediated through FAK.     

Primary dissociated cultures of human brainstem cells: a useful tool for their characterization and neuroprotection study

Dissociated cell cultures were prepared from brainstems of 5- to 10-week-old human fetuses. Catecholamine- as well as indolamine-containing cells were visualized using respectively dopamine (DA), noradrenaline (NA) and serotonin (5HT) as immunocytochemical markers. NA-, DA-, and 5HT-stained cells were characterized in the rhombencephalic cultures, representing respectively the fetal localization of the locus coeruleus and raphe nuclei. DA-stained cells were characterized in the mesencephalic cultures; these DA-cells originating from the substantia nigra presented morphological aspects different from the DA-rhombencephalic cells. Two types of GABA neurons and glial cells presenting glial fibrillary acidic protein (GFA-P) reactivity were also found in all the cultures. Two non-competitiveN-methyl-D-aspartate antagonists, 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) andcis-Pip/Me 1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine (GK11) in enantiomeric form (–), have been investigated for survival on rhombencephalic cultured cells. The number of 5HT-cells was found to be greater in the treated cultures than in the control ones. Thisin vitro system appears to be a useful tool for the investigation of the development of central nervous system (CNS) cells as well as the study of neuroprotection.Abbreviations CA catecholamine - DA dopamine - CNS central nervous system - GFA-P glial fibrillary acidic protein - IR immunoreactivity - NMDA N-methyl-d-aspartate - NA noradrenaline - 5HT serotonin - TCP 1-[1-(2-thienyl)cyclohexyl]piperidine - GK11 cis-Pip/Me 1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine     

Quantification of human neutrophil motility in three-dimensional collagen gels. Effect of collagen concentration.

Leukocytes must migrate through tissues to fulfill their role in the immune response, but direct methods for observing and quantifying cell motility have mostly been limited to migration on two-dimensional surfaces. We have now developed methods for examining neutrophil movement in a three-dimensional gel containing 0.1 to 0.7 mg/ml rat tail tendon collagen. Neutrophil-populated collagen gels were formed within flat glass capillary tubes, permitting direct observation with light microscopy. By following the tracks of individual cells over a 13.5-min observation period and comparing them to a stochastic model of cell movement, we quantified cell speed within a given gel by estimating a random motility coefficient (mu) and persistence time (P). The random motility coefficient changed significantly with collagen concentration in the gel, varying from 1.6 to 13.3 x 10(-9) cm2/s, with the maximum occurring at a collagen gel concentration of 0.3 mg/ml. The methods described may be useful for studying tissue dynamics and for evaluating the mechanism of cell movement in three-dimensional gels of extracellular matrix (ECM) molecules.     

Calcium regulation of mitochondria motility and morphology

In the Fifties, electron microscopy studies on neuronal cells showed that mitochondria typically cluster at synaptic terminals, thereby introducing the concept that proper mitochondria trafficking and partitioning inside the cell could provide functional support to the execution of key physiological processes. Today, the notion that a central event in the life of every eukaryotic cell is to configure, maintain, and reorganize the mitochondrial network at sites of high energy demand in response to environmental and cellular cues is well established, and the challenge ahead is to define the underlying molecular mechanisms and regulatory pathways. Recent pioneering studies have further contributed to place mitochondria at the center of the cell biology by showing that the machinery governing remodeling of mitochondria shape and structure regulates the functional output of the organelle as the powerhouse of the cell, the gateway to programmed cell death, and the platform for Ca²⁺ signaling. Thus, a raising issue is to identify the cues integrating mitochondria trafficking and dynamics into cell physiology and metabolism. Given the versatile function of calcium as a second messenger and of the role of mitochondria as a major calcium store, evidences are emerging linking Ca²⁺ transients to the modulation of mitochondrial activities. This review focuses on calcium as a switch controlling mitochondria motility and morphology in steady state, stressed, and pathological conditions.