RGD — Tutorials teaching on fibronectin

87 ment, and higher student ratings in college courses.' These results stem from several aspects of this teaching/ learning method which are also employed in Biology 411, viz, 'frequent quizzing, immediate feedback, a mastery requirement.' Keller, in his original paper describing the PSI method, 1 identified five features that distinguish PSI courses from conventional ones. PSI courses are: (1) mastery-oriented, and (2) individually-paced courses which use (3) a few lectures for stimulation and motivation, (4) printed study guides for communication of information, and (5) student proctors for quiz evaluation. A review 4 of PSI suggests that students learn and retain more in PSI classes '(a) when this progress is evaluated frequently using brief quizzes, (b) when they receive feedback immediately after each assessment, and (c) when they are required to re-do work until mastery is achieved.' Information on obtaining the teaching materials employed in this Introductory Biochemistry course may be obtained by writing to the author.

Acknowledgements The author is indebted to Mses Terri Tanaka and Alice Gembarski, previous proctors and superproctors, for their captious perusal of the manuscript, and Mses J. Boon Neo Ong, Lusiana Kartawidjaja, and Eileen Yu for their editorial skills and valuable suggestions. The following dedication appears in both the Unit Guides and the Proctor's Manual to this course: To my proctors, past, present, and future, without whom this way of passing knowledge of biochemistry would be impossible. To all of those 411 students willing to take a chance on learning biochemistry on their own, and without the tense push of competition.

References 1Keller, F S (1968)Journal of Applied Behavior Analysis 1, 79-89 2Kulik, J A, Kulik, C-L C and Smith, B B (1976) Journal of Programmed Learning and Educational Technology 13, 23-30 3Kulik, J A, Jaksa, P and Kulik, C-L C (1978)Journal of Personalized

Instruction 3, 2-14 4Kulik, J A, Kulik, C-L C and Cohen, P A (1979) American Psychologist 34, 307-318

Announcement 15th International Congress of Biochemistry Jerusalem, Israel, August 4-9, 1991 Every three years the advances in biochemistry are presented during the International Congresses of Biochemistry organized under the auspices of the International Union of Biochemistry. The 15th International Congress of Biochemistry will be held in Jerusalem, Israel, August 4-9, 1991. The Organizing Committee invites you most cordially to participate in this Congress. The early registration fee will be $340, and for young scientists (under the age of 30) $170. The first Circular with the pre-registration form is available from your local Biochemical Society or directly from the Secretariat of the- 15th International Congress of Biochemistry, KENES Ltd, P.O. Box 50006, Tel-Aviv 61500, Israel

BIOCHEMICAL EDUCATION 18(2) 1990

RGD m Tutorials Teaching on Fibronectin E J WOOD

Department of Biochemistry University of Leeds Leeds LS2 9JT, UK Introduction One aim of education is to prepare individuals to be able to function professionally over the next 30-40 years of their lives. In Biochemistry, professionals obviously need an extensive and flexible vocabulary and a good understanding of the major principles, models and investigative processes of the subject. They also need skills in problemsolving, the ability to find and use appropriate information, and the resilience that is essential if they are to cope with the changes in concepts, models and instrumentation that will occur during their working lives. The majority of teaching institutions have tended to 'give' more and more information to students and to reinforce the notion that this should be memorised more or less by rote, by asking for its recall, often in detail, in examinations. Writers of articles in this Journal have commented on this custom time and time again. I was reflecting along these lines when I found myself required to deal with the protein fibronectin as part of a course of 30 BSc Biochemistry students in their second year on the components of the extracellular matrix. Fibronectin is a very interesting protein from a number of viewpoints and my initial view was that students could hardly fail to be interested in it. However, on further reflection, it seemed to me that the topic was so information-rich, that, with the best will in the world, all that students could do, during a series of lectures on the topic, was to write down facts presented by the lecturer as fast as they could, without time for understanding and making connections. The letters R G D (arg-gly-asp) are associated with fibronectin, and it occurred to me that instead of giving a series of information-rich lectures, it would be possible to devise a Review, Get, Do approach with a view to turning the lecture time into an active, interactive, useful experience for learning and understanding. Here is a brief account of how it turned out.

R e v i e w what I already know: make connections

G e t information for a specific purpose (not for writing down undigested in the examination)

D o something: sitting in lectures listening is passive and is not conducive to learning

88 Background Fibronectins (FN) are high Mr glycoproteins that play a key role in various contact processes within vertebrates, including cell attachment, cell spreading, cell migration, control of cell morphology, differentiation and oncogenic transformation. The interactions are between cells and the extracellular matrix components. Fibronectins are also vital in embryological development and in wound healing. Fibronectins are dimers of subunits that are similar, but not identical, polypeptides of Mr about 240 000. The two polypeptides are joined at one end by disulphide bonds and along the chains, various domains may be distinguished having binding affinities for fibrin, heparin, collagen, cell surface and a number of other biological molecules such as DNA and Staph aureus protein A. Two major forms of FN can be distinguished: a cellular FN which is a component of the extracellular matrix of fibroblasts and other cell types, and a plasma FN which is a soluble form present at relatively high concentration in plasma (0.3 mg/ml) and probably has a role in opsonization, haemostasis and wound healing. Two other features of FN are important: (1) in the cellbinding domain, there occurs the sequence RGD, or ArgGly-Asp, which appears to be essential for binding, since peptides which contain this sequence inhibit binding; (2) the FN gene appears to be single and to contain about 48 exons, and which, by differential splicing of the primary transcript, can give rise to perhaps 20 different types of mRNA and consequently a family of polypeptides. Little is known as yet of how this is achieved or controlled, but no doubt this will change. Lecture Period 1 The students were given the 'blank' outline of fibronectin structure on a hand-out (Fig la). The same picture was shown on the overhead projector and gradually filled in with domain function and Mr by means of additional transparencies (Fig lb). At this stage time was taken to review students' knowledge of: heparin, fibrinogenfibrin, collagen, etc, by asking the students to speak

(a) K K K

K

K

K

K

briefly about these or to come prepared to talk about them for the next session. Later in this session the students were divided into groups of 3-4 to spend five minutes discussing what experiments might be carried out to isolate individual FN domains and to determine their functions. The suggestions were then shared with the class and discussed briefly. Although this activity generated the idea of using a short enzyme treatment, notions of actually how to carry out the experiment and to follow what was happening were rather vague. Lecture Period 2 After a brief presentation about the cell-binding (RGD) domain we reviewed the single letter abbreviations for the amino acids that occur in proteins and looked up in the Sigma catalogue what RGD-type peptides could be purchased. Then a discussion was held on how the cellbinding activity of fibronectin might be measured. This led into a consideration of cell culture methodology. In the latter half of this session the involvement of fibronectin in cell movement, cancer, embryology and wound healing were introduced. The students were divided into four groups and each group was given the assignment of finding out about one of these, each group cooperatively to prepare a poster for the following week. Literature references to some recent reviews were offered (see list at the end of this article). Lecture Period 3 To start with we had a look at the posters that had been prepared by the students. All groups had tried hard, but some were side-tracked and presented too much experimental detail on their posters. Nevertheless, the exercise stimulated attempts to look up the primary literature. We continued this session by looking at the gene for fibronectin. Firstly, we quickly reviewed transcription and translation, exons and introns, at a general level, and secondly the students read the article by Kornblihtt et al (1985) in the EMBO Journal aided by the diagram in the Scientific American article by Haynes (p 38). This was quite a good activity, although the students found reading the EMBO Journal article hard. It certainly illuminated and reviewed their ideas about eukaryotic RNA processing.

B

(b) 3K

30K He4xarin Fibrin Actin

40K

20K

Collagen Fibrin

3SK

3OK

75K

60K C¢II

Helxlrin

}~S

Fibrin

Figure 1 0 H P transparency showing fibronectin domain structure: (a) outline, (b) with all the information revealed BIOCHEMICAL E D U C A T I O N 18(2) 1990

Lecture Period 4 This was based around the piece b y Engvall in Current Contents (Fig 2) and was quite light-hearted. About five minutes were spent looking at and talking about Current Contents as a way of keeping up with the literature. A number of old copies were available for examination. We went on to talk about keeping abreast of the literature, library searches and the exponential growth of the literature. (This could have expanded to a discussion of why people publish, but it did not). We then took five minutes to read the piece by Engvall.

89

_This Week's Citation Classic"

CC/NUMBER 18 "'*',"" I

I EngvsllE & Ruo~d~fl E. Bindingof solubleform of fibroblastsurfaceprotein, |fibmnectin, to collagen./m.1. Cancer20:t-5, 1977. [Division of Immunololff, City of Hope N86omd Medk:zl Cemet. Duar~, CAI

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The paper shows that fibronectin binds to col- fromSigma.However,whenI got the collagen lagen, in particular to denatured collagen, or and was ready to do the experiment,which gelatin, and that fibronectincan be purified involved coating the microtiter wells with a using affinity chromatngraphy on gelatio-Seph~ solution of the collagen, I could not get the rose. [The 5CI• indicates that this paper has protein into solntion. been cited in over 1,315 pub[ications.] At that time we were running a lot of radioimmunoamays for tumor antigens, and the butused fm these assaysincluded gelatin (Knox gelatin) to reduce nonspacific interactions and background, tt dawned on me that gelatin had Interactions with Fibronectins something to do with collagen, and I used the radioimmunnassay buffer to coal some wells Eva Engvall in my assay plate. The result ol the assay was La Jolla Cancer Research Foundation spectacular. FibronecUn appeared to bind ex10901 North Torrey Pines Road ceptionally well to gelatin and much less well, La Iolla, CA 92037 if at all, to native collagen. It was hard to convince my colleagues, though, that this was a December 22, 1988 specific binding of biological relevance. But aher I had completed a purificallun of fibronecfin from plasma using affinity chroThis work was started soon after Erkki matography on gelatin coupled to SephaRunslahti and t had moved to the City of Hope ruse--with unparalleled speed and yield of reMedical Center in Duarte, Cafifomla, horn the covery-there was no doubt about the speciUniversity of Helsinkl, Finland. Erkkt was one ficily, and the practical significance of the of the discoverers of "SF antigen" (surface-of finding was obvious. Nevertheless, to this day fibroblast antigen), later renamed "fibronnc- the biological significance of the binding of tin." I was a posldoc. One of my projects was fibroneclin 1ogelatin rather than to the native to study possible interactions of fibronectin collagen is not clear. with other proteins. It was already known that Even eating huge quantities of jello does not fibronectin bound weakly to fibrinugen and seem to affect the circulating fibronectin levfibrin I and that it was present in connective els, We did that experiment, The purification tissue and extracellular matrices, possibly be- of fibronecfin using the affinity chromatogracause it was bound to another extracellular phy described in this paper was of great help protein. I was using a modified ELtSA2 to in fibronec~in research, 3 and for this reason study the interaction of fibrunectin with other the paper has been cited often. On the other proteins, and the assay worked relatively well hand, because it was so easy to modify the for the fibrinogen-fibronectin interaction when technique by using different agents fo~ elution, fibrinogeo-coated microtiter wells were used nsany other workers have published their own for binding and enzyme.labeled antifibronoc- off-cited papers and. our paper has not been tin was used for detection, t then decided to cited as often as the melhod has been used. test whether fibronectin might bind to collaIt is exciting to have been participating in gen. I had very little knowledge about collagen the virtual explosion of exlracellular matrix reat that time other than that it was an abundant search in the la~ 10 years.4 II is also reconoective-tissue protein with a ropelike struc- wardingto thinkthat someof the methodswe ture and that it was available for purchase developed helped.

I. UumlaaU E A Vttherl A. Imate~ee of soluble flbmEtmt surface tmtisemwith fib~nogen tnd fibria: identity with cold insoluble sloE*aBeof hmmm plasma.I. E~. Maff. 141:497-501. 1975. (Cited 3S0 timel.) 2. ruqtvall E & l~.rbual~ IP. Enzym~-linkmlimmtovt~otbemut~/. EUSA I~. Q~umtl~tionof tp~ifl¢ amtibodia by ¢l~ymelabeled anti-immunoBlobulthin imtigc~Cmledrebel I. /mmum~eay109:129-35. 1972, (Cited 1.900 tim~,) [See idto: E/qlvldl It'. Citadoa CIItSIiC,Cu.'mm'Coatcms/Lffe ~ 3Oil2):lS. 23 Mmch 1987.I 3. Uuethhtl E, Enltv~ E • Ihyuum g O. F i ' ~ , ~ a : ctmmetcxmoe~ of ~ tm~ture and func'tiom. Co~NrenReg. 1:95-128, 19Sl. {Cited 595 tlmet) [See the: RtmsblthU![. Citafioe Cla/slc. ~ Come,eti/C2i~at/MecL~iae 17(20). 15 May 19119imd CC,T..ffe.f~enc~ 32(201. I$ May 19119. la !tm~.I 4. nueldahtl K • IPktt'/chbecberM D. New pmpectlves in cell edh~doe: RGD u d [l~lfglfi111.~ 23S:491-7, 19rL

Figure 2 Current Contents, Citation of the Week, by E Engvall, reproduced by kind permission of the Institute[or Scientific Information

Several questions were asked and led to discussion. The main ones were: (1) What is ELISA? (2) What is gelatin? (3) How was the affinity purification method carried out? These topics have rather little to do with fibronectin directly but they permitted a great deal of teaching to be done on immunology, collagen structure, affinity chromatography and protein purification. I felt that this teaching and discussion was more fruitful and indeed more enjoyable for the students because they wished or needed to know about these things for a purpose, namely to understand fibronectin. This 'need to know' aspect made the activity seem worthwhile, and was very different from presenting the 'facts' in a lecture on each of these topics. B I O C H E M I C A L E D U C A T I O N 18(2) 1990

Comment Although a formal questionnaire was not used, the students appeared to enjoy the activities, if only as a complete, and sometimes daunting, change from their more normal activity of being lectured to. As the teacher, I felt that we had established good rapport and that the students had made many connections and gained an insight into how biochemists operate in their daily lives. We had explored the literature and the problem of keeping up with it. The students appeared to understand that the reasons for wanting facts, and therefore for searching for them in the literature, was in order to comprehend something, to find out about experiments that had carried out and to obtain a basis for doing their own experiments in the laboratory. Many of the activities were carried out on a cooperative group basis rather than a competitive individual one. Next year I shall do some of the things slightly different in the light of experience. This may include giving more advance notice of assignments and getting the students to write a summary of a short paper from which the Abstract has been removed. Probably we will use a questionnaire at the end too. It occurred to me that almost anything might be taught in this w a y . . . Literature General Reviews Alberts, B et al 'Molecular Biology of the Cell' (second edition), Garland, New York, pp 816-818 Haynes, R O (1986) Fibronectin, Scientific American 254, 42 Ruoslahti, E (1988) Fibronectin and its receptors, Ann Rev Biochem 57, 375 Stryer, L (1988) 'Biochemistry' (third edition), W H Freeman, New York, pp 277-278 Yamada, K M (1989) Fihronectin - - structure, function and receptors, Current Opinion in Cell Biology I, 906

Specific Papers Kornblihtt, A R et al (1985) Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene, EMBO J 4, 1755 Maeda, T et al (1989) A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide, J Biol Chem 264, 15 165 Pierschbacher, M D and Ruoslahti, E (1984) Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule, Nature 305, 30 Thiery, J P (1980) Fibronectin in early embryogenic development of the vertebrate, BioEssays 2, 32 Adams, J C and Watt, F M (1989) Fibronectin inhibits the terminal differentiation of human keratinocytes, Nature 340, 307 Cheng, C Y e t al (1988) Fibronectin enhances healing of excised wounds in rats, Archives o[ Dermatology 124, 221 O'Keefe, E J e t al (1985) Spreading and enhanced mobility of human keratinocytes on fibronectin, J Investigative Dermatology 85, 125 Saga, S et al (1988) Enhanced fibronectin expression in Rous sarcoma virus-induced turnouts, Cancer Research 48, 5510

Acknowledgement I am very grateful to Alan Mehler and Frank Vella for reading this manuscript and making helpful comments.