H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 98
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The extracellular regions in all these proteins containmultiple epidermal growth factor (EGF) repeats, which maydirectly participate in binding to other molecules. SomeWAKs have been shown to bind to glycine-rich proteins inthe cell wall, thereby mediating membrane–wall contacts.These Arabidopsis proteins have a single transmembrane domain and an intracellular cytosolic tyrosine kinase domain,which may participate in signaling pathways somewhat likethe receptor tyrosine kinases discussed in Chapter 14.The results of in vitro binding assays combined with invivo studies and analyses of plant mutants have identifiedseveral macromolecules in the ECM that are important foradhesion.
For example, normal adhesion of pollen, whichcontains sperm cells, to the stigma or style in the female reproductive organ of the Easter lily requires a cysteine-richprotein called stigma/stylar cysteine-rich adhesin (SCA) anda specialized pectin that can bind to SCA (Figure 6-35).Disruption of the gene encoding glucuronyltransferase 1,a key enzyme in pectin biosynthesis, has provided a strikingillustration of the importance of pectins in intercellular adhesion in plant meristems. Normally, specialized pectin molecules help hold the cells in meristems tightly together. Whengrown in culture as a cluster of relatively undifferentiated cells,called a callus, normal meristematic cells adhere tightly andcan differentiate into chlorophyll-producing cells, giving thecallus a green color.
Eventually the callus will generate shoots.In contrast, mutant cells with an inactivated glucuronyltransferase 1 gene are large, associate loosely with each other, anddo not differentiate normally, forming a yellow callus. The introduction of a normal glucuronyltransferase 1 gene into themutant cells by methods discussed in Chapter 9 restores theirability to adhere and differentiate normally.The paucity of plant adhesive molecules identified todate, in contrast with the many well-defined animal adhesivemolecules, may be due to the technical difficulties in workingwith the ECM/cell wall of plants.
Adhesive interactions areoften likely to play different roles in plant and animal biology, at least in part because of their differences in development and physiology.KEY CONCEPTS OF SECTION 6.6Plant TissuesThe integration of cells into tissues in plants is fundamentally different from the assembly of animal tissues, primarily because each plant cell is surrounded by a relativelyrigid cell wall.■▲ EXPERIMENTAL FIGURE 6-35 An in vitro assay used toidentify molecules required for adherence of pollen tubes tothe stylar matrix. In this assay, extracellular stylar matrixcollected from lily styles (SE) or an artificial matrix is dried ontonitrocellulose membranes (NC).
Pollen tubes containing spermare then added and their binding to the dried matrix is assessed.In this scanning electron micrograph, the tips of pollen tubes(arrows) can be seen binding to dried stylar matrix. This type ofassay has shown that pollen adherence depends on stigma/stylarcysteine-rich adhesin (SCA) and a pectin that binds to SCA. [FromG. Y. Jauh et al., 1997, Sex Plant Reprod.
10:173.]The plant cell wall comprises layers of cellulose microfibrils embedded within a matrix of hemicellulose,pectin, extensin, and other less abundant molecules.■Cellulose, a large, linear glucose polymer, assemblesspontaneously into microfibrils stabilized by hydrogenbonding.■The cell wall defines the shapes of plant cells and restricts their elongation. Auxin-induced loosening of the cellwall permits elongation.■6.7 • Growth and Use of Cultured CellsAdjacent plant cells can communicate through plasmodesmata, which allow small molecules to pass betweenthe cells (see Figure 6-34).■Plants do not produce homologs of the common adhesive molecules found in animals.
Only a few adhesive molecules unique to plants have been well documented to date.■6.7 Growth and Use of Cultured CellsMany technical constraints hamper studies on specific cells orsubsets of cells in intact animals and plants. One alternative isthe use of intact organs that are removed from animals andperfused with an appropriately buffered solution to maintaintheir physiologic integrity and function. Such organ perfusionsystems have been widely used by physiologists. However, theorganization of organs, even isolated ones, is sufficiently complex to pose numerous problems for research on many fundamental aspects of cell biology.
Thus molecular cellbiologists often conduct experimental studies on cells isolatedfrom an organism and maintained in conditions that permittheir survival and growth, a procedure known as culturing.Cultured cells have several advantages over intact organisms for cell biology research. First, most animal and planttissues consist of a variety of different types of cells, whereascells of a single specific type with homogeneous propertiescan be grown in culture. Second, experimental conditions(e.g., composition of the extracellular environment) can becontrolled far better in culture than in an intact organism.Third, in many cases a single cell can be readily grown intoa colony of many identical cells, a process called cell cloning,or simply cloning (Figure 6-36).
The resulting strain of cells,which is genetically homogeneous, is called a clone. This simple technique, which is commonly used with many bacteria,yeasts, and mammalian cell types, makes it easy to isolate genetically distinct clones of cells.(a)(b)▲ FIGURE 6-36 Cultured mammalian cells viewed at threemagnifications.
(a) A single mouse cell attached to a plastic petridish, viewed through a scanning electron microscope. (b) Asingle colony of human HeLa cells about 1 mm in diameter,produced from a single cell after growth for 2 weeks. (c) After235A major disadvantage of cultured cells is that they arenot in their normal environment and hence their activitiesare not regulated by the other cells and tissues as they arein an intact organism.
For example, insulin produced by thepancreas has an enormous effect on liver glucose metabolism; however, this normal regulatory mechanism does notoperate in a purified population of liver cells (called hepatocytes) grown in culture. In addition, as already described,the three-dimensional distribution of cells and extracellularmatrix around a cell influences its shape and behavior.
Because the immediate environment of cultured cells differsradically from this “normal” environment, their propertiesmay be affected in various ways. Thus care must always beexercised in drawing conclusions about the normal properties of cells in complex tissues and organisms only on thebasis of experiments with isolated, cultured cells.Culture of Animal Cells Requires Nutrient-RichMedia and Special Solid SurfacesIn contrast with most bacterial cells, which can be culturedquite easily, animal cells require many specialized nutrientsand often specially coated dishes for successful culturing. Topermit the survival and normal function of cultured tissues orcells, the temperature (37 °C for mammalian cells), pH, ionicstrength, and access to essential nutrients must simulate asclosely as possible the conditions within an intact organism.Isolated animal cells are typically placed in a nutrient-rich liquid, the culture medium, within specially treated plastic dishesor flasks.
The cultures are kept in incubators in which the temperature, atmosphere, and humidity can be controlled. Toreduce the chances of bacterial or fungal contamination, antibiotics are often added to the culture medium. To furtherguard against contamination, investigators usually transfercells between dishes, add reagents to the culture medium, andotherwise manipulate the specimens within special cabinets(c)cells initially introduced into a 6-cm-diameter petri dish havegrown for several days and then been stained, individual coloniescan easily be seen and counted. All the cells in a colony areprogeny of a single precursor cell and thus genetically identical.[Part (a) courtesy of N.
K. Weller. Parts (b) and (c) courtesy of T. T. Puck.]236CHAPTER 6 • Integrating Cells into Tissuescontaining circulating air that is filtered to remove microorganisms and other airborne contaminants.Media for culturing animal cells must supply histidine,isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine; no cells in adult vertebrate animals can synthesize these nine essential amino acids. Inaddition, most cultured cells require three other amino acids(cysteine, glutamine, and tyrosine) that are synthesized onlyby specialized cells in intact animals.
The other necessarycomponents of a medium for culturing animal cells are vitamins, various salts, fatty acids, glucose, and serum—the fluidremaining after the noncellular part of blood (plasma) hasbeen allowed to clot. Serum contains various protein factorsthat are needed for the proliferation of mammalian cells inculture. These factors include the polypeptide hormone insulin; transferrin, which supplies iron in a bioaccessibleform; and numerous growth factors.
In addition, certain celltypes require specialized protein growth factors not presentin serum. For instance, hematopoietic cells require erythropoietin, and T lymphocytes require interleukin 2 (Chapter14). A few mammalian cell types can be grown in a chemically defined, serum-free medium containing amino acids,glucose, vitamins, and salts plus certain trace minerals, specific protein growth factors, and other components.Unlike bacterial and yeast cells, which can be grown insuspension, most animal cells will grow only on a solid surface.
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