MORPHOLOGICAL STUDY OF MEMBRANE BEHAVIOR DURING REDIRECTIONAL MIGRATION IN HUMAN FIBROBLAST CELLS
Carol A. Heckman, Advisor
Cell migration has been studied for half a century to reveal the mechanisms of related life and multicellular activities like embryogenesis in development biology, tissue invasion and tumor metastasis in oncology, biomedical material development in biomedical engineering, and some other biological fields.
Cell migration could be recognized by several steps. Two membrane protruding structures, filopodia and lamellipodia, locate near the front edge providing the protruding force to the cell migration and lying down the focal adhesions behind to fix the cell on the substrate and gain contractile force from the myosin-actin interaction. The release of focal adhesions near the lagging edge leads to the detachment of the rear end then disrupts the cell-substrate conjunction. However, the cell reaction to obstacle during the migration has been rarely studied. In this research, a boundary between a preferred substrate and a non-preferred one was created by sputtered germanium on a plastic culturing surface. Nano-gold particles were applied for migration speed and directional persistence tracking. The interaction between the cells and the boundary studied with scanning electron microscope (SEM) revealed the according changes of membrane structures resulted from cell redirecting at the edge of non-preferred substrate. The shape analysis of cell includes the their edge being traced with Photoshop and computerized by program to synthesize the statistical data showing the morphological features of cell membrane changing throughout the migration redirection process.
This is a research of a bold idea and great creativity in methology on cell migration; it revealed the morphological features related to cell random walking on a uniformed or a gradient substrate, which is meaningful to understand cell contact inhibition related problems in tumor metastasis and its therapeutic treatments.
Cell migration is a common but important cell behavior relating to development, diseases, and maintaining the stability in multicellular organisms. Cell migration has been demonstrated driving many development stages in embryogenesis by cell specification, hindgut expansion, gastrulation, early gonad formation, and some other embryonic development processes. Besides being helpful to understand the early embryogenesis, another reason that cell migration has been studied is its therapeutic role in cancer biology and related drug designing. Tissue invasion is a crucial step in the tumor metastasis and has been demonstrated closely related to cell migration by actin dynamics. In tumor cells, bundled or densely arrayed actins filament form filopodia and lamellipodia on the cell leading edge during migration helping the invasion process with the presence of matrix degradation (Vignjevic & Montagnac, 2008). Additionally, neurogenesis and neuron development are closely linked with cell migration. Neuron cells have to elongate from their birth site with the facilitation of cytoskeleton construction. Failure in migration might lead to severe neuron development disorder related diseases (Gleeson & Walsh, 2000). In any multicellular organism, the sustenance of system stability is undoubtedly important; wound healing is one example. Once a wound is created on an epithelial cells covered surface, the cells will have the potential to close the gap through cell migration and cell proliferation, which is meaningful to the maintenance of any living system, cell migration research, and biomaterial application in tissue engineering (Tremel et al., 2009).
The migration process is initialed by the polarization, which could be induced by applying microscopic nonuniformities or changing the receptor-ligand binding kinetics. Polarization determines that the cell has its front and rear regions during the migration process, which is earmarked by the membrane protruding structures activities. There are two kinds of membrane protruding structures which could be found near the cell front edge. One is the needle-like filopodia, regulated by cdc42, and the other is the sheet-like lamellipodia regulated by rac. The formation of these two kinds of protrusions is the consequence of local actin polymerization and crosslink which provides the protrusive force for the migration process. The other cell translocation force helping the migration process is the contractile force, which is believed to be related to the myosin-actin interaction. A third membrane structure playing a key role in providing translocation force is the focal adhesions. Small new focal adhesions regulated by rho, which is initialed by the activation of cdc42 form behind the lamellipodia and fix to the substrate. It is believed that the formation of the adhesive complex is comprised by phosphoprotein covalently modified by tyrosine phosphorylation. The detachment of the focal adhesions near the rear edge will disrupt the cell-substrate conjunctions, which release the cell from the reaction of the substrate.
Since cell behavior is closely related to the features of various membrane structures, quantification and classification of those features help to interpret cell behavior for a better understanding of the membrane behavior during the cell migration. Our laboratory established a program to quantify and category the graphic trace line information of a migrating cell into different factor numbers with various values according to their significance throughout the peripheral cytoplasm of the cell. There are 20 latent factors yielded by the analyzing program, which could be used to identify and describe specific features on cell phenotypes. Among the 20 factors, four of them are predominantly corresponded for evaluating cell edge and protruding features. Factor 4 stands for the size and the percentage of filopodia or microspikes; factor 7 characterizes the features which is broader and bulkier than filopodia; factor 5 represents long, sprawling protrusions or deep indentations; factors 16 denotes the tapering projections which is larger than filopodia and also deeply anchoring in the cytoplasm.
Besides the standard quantification method illustrating the membrane features during the cell migration, the track information symbolizing the cell behavior which is the other approach in my study understanding the correlation between them could be quantified by two indexes, linear cell locomotion speed and directional persistence time, which are the description or the measurement of the migration speed and its consistency of migrating direction. Usually, the linear cell locomotion speed inversely correlates with the contractile force. The coordination between the directional signal and the physical movement leads to the inverse correlation between the migration speed and the persistence of the moving direction. Because the membrane protrudes near the cell front, the cell then changes its walking direction randomly, which is named random walking (Lauffenburger & Horwitz, 1996).
Though cells could wander randomly on the substrate when certain inducements are applied, the two indexes mentioned above could be controlled on some extent for specific studies. A series of research demonstrate the linear cell locomotion speed could be manipulated by using various growth factors. Platelet-derived growth factor (PDGF-BB) has been found to promote the bone tissue injury in vivo and to have significant healing effect in the ‘scratch’ assay in vitro (Chung et al., 2009). More research showed the insulin-like growth factors could stimulate the tissue invasion and tumor metastasis by promoting cell migration (Guvakova, 2007). In lung cancer research, it was suggested that the interaction between the ion channel TRPM7 and the epidermal growth factor (EGF) was required for tumor development related cell migration (Gao et al., 2011). The directional persistence could be related to the cell sensory mechanism. It has been demonstrated that the filopodia has been used by cells to sense the external environment (Mellor, 2010). Furthermore, evidence showed the small adhesions formed in the filopodia focal complex (filopodial FX) right behind along the filopodia axis. When lamellipodia substitute those regions, those small adhesions get matured into classic focal adhesions. The filopodia then could elongate again for further migration. This evidence suggested that the filopodia are responsible for the sensory mechanism in the cell migration (Schäfer et al., 2009). More evidence on the molecular level is needed for further explanation of the correlation between the filopodia formation and the protein composition depends on the co-culturing medium and gradient substrate.
To study the morphology of cell re-orientation during migration, durotaxis provides a very important breakthrough into the question to help understand the connection between the mechanical and chemical effectors on this process.
Cell migration could be regulated by the chemicals in the culturing medium and mechanical environmental factors. Durotaxis is a phenomenon whereby cells can sense the substrate rigidity in a gradient, and then change their migration speed and direction accordingly. In some earlier experiments, the cells showed a strong attachment to stiff surfaces like plastic and glass. Then, people started to reveal the correlation between the stiffness of the substrate on which cells grow and the cells’ preference. The results from the former research particularly showed that cells can hardly form adhesions and thus cannot keep steady on a soft substrate, while on the same time they have difficulty to grow on very rigid substrate because the adhesions cannot be released from the surface. Briefly, the researchers found that a substrate with an intermediate stiffness is most preferred by cells so that the cells can efficiently grab the surface at their leading edge and detach the rear, which is known as two essential steps affecting the cell migration indexes including speed and orientation.
To see how cells react to the substrates, soft and rigid, differently, a boundary of these two substrates was created and the according cell migration trajectory was studied. When migrating from the soft to the stiff side of the substrate, cells passed the boundary readily, while coming from the opposite direction but the same step in rigidity; cells approached the boundary with a certain angle and changed their original migrating direction to move along the boundary. Noticeably, a critical angle was coined when it was discovered that the cell might make mistakes crossing the boundary to the soft side when the approaching angle was between 43.2o and 90o. A solid’s elastic modulus E was applied to describe the stiffness of the substrates during the study. The relative change in the substrate by the applied force could be obtained as the value of module E or another common method, pocking the substrate with macro- and micro-indenters by using atomic force microscopes (AFM), could also be applied to measure the module E.
In my research, the boundary between the two substrates, plastic and metal, of different stiffness distinguishedly interfered cell migration process with the stimulation of migration rate by various growth factors. The tracking and tracing information together elucidated the correlation between the cell behavior and the membrane protruding features proved to be closely related to upstream singling pathway and series of biochemical reactions, which would point out a way for further biochemistry and molecule study and eventually contribute our understanding of cancer by making the cell migration process controllable.
2. MATERIALS AND METHODS
2.1Cell and cell culture
To make 2 L of the medium, 20.0 g Dulbecco’s Modified Eagle Medium (Gibco, Grand Island, NY) powder was initially dissolved in 1600 ml deionized water combined with 20 ml of penicillin-streptomycin solution (Gibco, Grand Island, NY) and 11.2 ml of fungizone amphotericin B (Invitogen, Paisley, Scotland, UK). 1N NaOH (Fisher Chemicals, Fair Lawn, New Jersey) was used to adjust the pH of the solution to neutral when color of the solution changed from golden to ruby. The final volume of the no serum DMEM solution was supplemented to 1800 ml with deionized water for a 200 ml volume reserved for the addition of Fetal Bovine Serum (FBS). The solution was filtered with a sterilized Millipore filter of 90 mm in diameter and 0.22 um in pore size (Millipore, Billerica, MA) and collected in 4 sterilized 500-ml glass bottles, 450 ml in each. For subculturing, 50 ml of FBS (HyClone, Logan, Utah) was added into each bottle for a 1% of serum concentration while some part of the no-serum medium was kept for experimental study. A small amount of medium from each bottle was tested for sterility before applied on cell culturing.
A Trypsin-EDTA HBSS was prepared for dissociating subcultured cells from the old dishes. To make a solution, 50 ml of 10X HBSS (Gibco, Grand Island, NY), 0.875 g Na-EDTA, and 100 ml of deionized water were dissolved in a big flask. On the other hand, 10 mg phenol red (Matheson, Cincinnati, OH) and 175 mg NaHCO3 (Matheson, Norwood, OH) were dissolved in deionized water until reached a 400 ml volume before combined with the solution in the big flask. To adjust the pH, 1N NaOH was added into the solution until the liquid obtained a cherry red color. Deionized water then was added into the solution to reach a final volume of 437.5 ml. The solution was autoclaved before being separated into 5 100-ml glass bottles, 87.5 ml to each. Before applied on cultured cells, 12.5 ml of 0.255 1X Trypsin (Gibco, Grand Island, NY) was added into each bottle, and then the optimal incubating time was tested based on specific cell line.
Human fibroblast cells (GM21808, Coriell Institute for Medical Research) of circumcised newborns foreskin purchased from NIH were cultured with DMEM medium with a supplement of 10% FBS (HyClone, Logan, Utah), dissociated from the old culturing surface by incubating with the Trypsin-EDTA HBSS for 4 minutes, and collected by centrifugation at 740rpm for 5 minutes. The cells were cultured with the medium in an incubator at 37 ℃ with 5% CO2 and 95% air. A sterilized glass tray containing distilled water was kept on the bottom rack of the incubator for humidity.
2.2Creating the boundary between two different substrate
The petri dishes were cleaned in pure ethanol overnight and were wiped clean on lens paper. 1/16” slim tape was used to partially cover the bottom of the culture dishes with 1/16” in between. The plastic petri dishes with the slim tape were coated with germanium in the Danton Sputter Coating Machine. Before loading the sample and the metal into the coating machine, the whole working area on the machine should be wiped with ethanol to eliminate the contamination of grease and remains. Also, the old filter was changed to avoid the oil diffused from the pump preventing a tight conjunction between a very thin layer of germanium the petri dish plastic. After the machine was settled and the petri dishes were ready for coating, the basket was loaded in the coating chamber with 0.02g germanium each time while the prepared petri dishes were fixed on the coating plate, four at a time. After the vacuum level in the bell chamber reached to 2×10-5 Pa, the coating plate rotating speed was set at 10% of full speed for an average coverage. The high voltage button then was released, and the output power was gradually raised until the voltage reached 42~45 Kv and 9 amp approximately. The amount of the remaining germanium in the wire basket was checked through a piece of dark glass to manipulate the coating process of the petri dishes, and the coating was completed only when the petri dishes obtained an even, shiny, dark brown coating. High voltage and rotating power were cut off and the chamber was vented with compressed air until it reaches the atmosphere. The coated dishes were obtained when the chamber was opened.
2.3Making cell migration tracking system
1% BSA (Sigma-Aldrich, St. Louis, MO) was kept at 4℃ overnight or filtered with a syringe loaded the Millipore filter of a pore-size of 0.22 um for instant use to obtain a complete dissolving. First, the slim tape was peeled from the petri dishes to create germanium-plastic boundaries before the 1% BSA was poured into the dishes. After the dishes were emptied by a pump and dried for 5 seconds, the pure ethanol was poured on the dishes to denature the BSA for an optimal adhesiveness for the nano gold particles. After the dished were emptied with a pump again, a dryer was applied to raise the surface temperature of the dishes to 90℃ in order to tightly stick the layers of protein, germanium, and the plastic dishes to avoid exfoliation in water based medium during the follow steps. After a mixture of 1.8ml of mM 14.5 AuCl4H (Alfa Aesar, Ward Hill, MA), 6ml of 36.5mM Na2CO3(Fisher Chemicals, Fair Lawn, New Jersey),and 11 ml of distilled water were boiling in the flask on a hot plate, 1.8 ml of 0.1% paraformaldehyde (Polysciences, Warrington, PA) was added into the boiling mixture. The solution was ready for use when boiling again and obtaining a muddy dark-blue to dark purple color. The still hot nano gold particles solution was poured onto the prepared petri dishes topped with germanium and denatured BSA. The dished were opened for a short while to avoid steam building up in under the lids. The dishes were then closed and kept in the incubator under the cell culturing condition for at least 45 minutes to let the nano-gold particles settle down. The solution was replaced with the culturing medium afterwards and kept in the incubator overnight. The prepared dishes were gently rinsed in PBS to eliminate the paraformaldehyde remains on the nano-gold particles coated surface. The dishes were kept with PBS buffer at 4℃ for later use within two weeks.
2.4Medical treatment for improving cell motility on the substrate
412.5 ml of 2ng/ml Dexamethasone (Sigma-Aldrich, St Louis, MO), 1.5ml of 1% Human Serum Albumin (HSA) (Sigma-Aldrich, St Louis, MO), and 30ul of 10ng/ml Platelet-Derived Growth Factor-BB (PDGF-BB) (Sigma-Aldrich, St Louis, MO) were added into every 15ml of no-serum DMEM medium. The cells collected from the old subculturing dishes to the prepared medium with the medicine treatments were plated on the prepared petri dish at a density of 5000 cell/dish. The cells were incubated for 48 hours.
2.5Microscopy for track and trace
The dishes with the cell samples were emptied and fixed in 3% paraformaldehyde for 10 minutes, then rinsed in 0.1M PBS buffer for 2 times. The sample dishes were observed with PBS buffer with a phase contrast inverted optical light microscope. The cells and their tracks near the germanium boundary are photographed and numbered for track study.
The sample dishes were refixed in a 2.5% glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.2, for 30 minutes. After rinsed with phosphate buffer 2 times, 15 minutes each, the dishes went through a s graded series of ethanol: 40%, 60%, 80%, 95%, 100%, 100%, 100%, 15 minutes each, for dehydration. When finished with the dehydration step, the walls of the dishes were removed and the interested bottoms carrying cell samples were trimmed to fit into the well in the critical point dryer. The dehydrated cell samples were coated with 2 nm of palladium ,mounted on aluminum stubs with colloidal graphite(Electron Microscopy Sciences, Hatfield, PA), and kept in a desiccator overnight for SEM photographing. Each of the cells was taken several pictures at certain magnification to show the edge of cells clearly on the photos.
2.6Shape analysis of track and trace
The track images of the cells were loaded on the ArcGIS program for shape analysis. Proper coordinate systems were established on the tiff image for following the measurement. Polygons and polylines were determined by the edge of the tracks; the former features were made for extracting the parameter and area while the later were for synthesizing Euclidean distance from the edge into the polygonal track patterns for objectively defined central lines of migration routes from the original images. The lengths of the lines were measured in ArcGIS and the orientation of the cell to the boundary will be measured with the IMAGE J program for angular data.
For trace information, the TIFF pictures with a resolution of 1024×768 from one cell were put together in the Photoshop program and two transparent layers were created to manually trace the cell leading edge and trailing edge. The trace layers were flattened separately from the background and trimmed to accommodate the program for the objective analysis of the cell membrane features by cataloging them with different effector numbers.
Variables were analyzed separately aligned in one block of the Best Subsets Regression while all factors including all the cell top and bottom information were aligned in the other block. The best subsets for the following regression were evaluated and selected. P value <0.05 determined that correlated factor was significantly related to the variable.
3.1Filopodia at the front edge of cell and the large tapering projection at the trailing edge of the cell showed high correlation to the cell membrane recovery after turning
F4t (factor 4 value of cell top), f4b, f5t, f5b, f7b, and f16b were selected from the Best Subsets Regression analysis showing their contribution to the regression. The following regression analysis showed that the f4t and f16b were of high significance to the correlation. P value of f4t was 0.028 while that of f16b was 0.007, showing the filopodia or the microspike structures at the front edge of the cell and the large tapering projection at the trailing edge of the cell were appreciably outstanding while cell was migrating away from the turning point.
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