Let's Not For­get Ac­etab­u­laria

by Elio

If I asked you what was the ex­per­i­men­tal ba­sis for the cen­tral dogma of bi­ol­ogy (DNA makes RNA makes Pro­tein), you would be likely to men­tion the clas­si­cal find­ings that the trans­form­ing prin­ci­ple was DNA (Av­ery et al.) or that phages trans­fer DNA to the host (Her­shey & Chase). How­ever, it is un­likely that you even have heard that the pre­cept was ear­lier de­rived from stud­ies with a uni­cel­lu­lar ma­rine alga, Ac­etab­u­laria. If so, you would miss the re­mark­able bi­ol­ogy that made it pos­si­ble to carry out this work. Here is why: Ac­etab­u­laria is such a large cell that it can be read­ily han­dled with one's hands.  It can be am­pu­tated into pieces that can be grafted to­gether and its nu­cleus trans­planted as eas­ily as walk­ing in the park.

Fig­ure 4. A clump of Ac­etab­u­laria ac­etab­u­lum. Source

Most cells are clearly too small for such lux­u­ries. To en­joy them, we must turn to the out­liers in range of sizes, that is, to gi­ant cells. So, how big can cells get? The cham­pion seems to be an­other a ma­rine alga, Caulerpa, which can reach 3 me­ters in length. It is mult­i­n­u­cle­ated, which seems al­most like cheat­ing (con­sider acel­lu­lar slime molds, which can also reach enor­mous sizes, and other coeno­cytic or­gan­isms). In­ci­den­tally, Cauler­pas are ed­i­ble and are called sea grapes in Ok­i­nawa (海葡萄 or umi-budō). Also mult­i­n­u­cle­ated are the xeno­phyophores, foraminifera-like pro­tists that live in the ocean at depths be­low 500 me­ters and reach 15 cm across (and which were men­tioned here ear­lier). Among the largest un­in­u­cle­ated sin­gle cells are the foraminifera called Num­mulites, which can reach 5 cm in di­am­e­ter, and a ma­rine ameba called Gro­mia spher­ica. But the gi­ant-celled al­gae, in­clud­ing Ac­etab­u­laria, are high on this list. Of course, birds' eggs are even larger and they are uni­cel­lu­lar and un­in­u­cle­ated all right, but their con­tents are more suit­able for an omelet than for ex­per­i­men­tal ma­nip­u­la­tions.

Fig­ure 2. The hap­loid and diploid por­tions of the life cy­cle of A. ac­etab­u­lum. Por­tions of the life cy­cle that com­prise a unit of de­vel­op­ment, i.e., all of ga­me­to­ge­n­e­sis, are united by an arc. Car­toons of the or­gan­ism are not to scale. Ad­di­tional de­tails about each por­tion the life cy­cle, in­clud­ing the rel­e­vant sizes of each por­tion of the life cy­cle, can be found in the text. Source

But first, what are Ac­etab­u­lar­ias ?   They are um­brella-like green al­gae called — prob­a­bly by some Vic­to­rian era nat­u­ral­ist — "mermaid's wine glasses" that are found in sub­trop­i­cal seas. They will re­mind you of the leaves of nas­tur­tium, should you re­mem­ber what these look like (en­tirely round leaves atop cen­tral stems). Ac­etab­u­lar­ias can reach an as­tound­ing 3 − 6 cm in height and con­sist of a slen­der stalk that is usu­ally at­tached to a rock sur­face by a rhi­zoid and which ends in a lo­bate um­brella-like cap. A large nu­cleus (50 − 120 μm in di­am­e­ter) is lo­cated at the rhi­zoid. This nu­cleus di­vides re­peat­edly as the alga ma­tures and the daugh­ter nu­clei are car­ried up­ward by cy­to­plas­mic stream­ing to end up in each of the lobes of the cap.

What Made Them Fa­mous?   Ac­etab­u­lar­ias were used in the early days of cell bi­ol­ogy for some amaz­ing ex­per­i­ments on nu­clear trans­plan­ta­tion and cel­lu­lar de­vel­op­ment. About 75 years ago, Joachim Häm­mer­ling dis­cov­ered that he could cut the or­gan­ism in half with im­punity and that each half would re­grow the alga. He also found that if he cut it again, the spec­i­men that had come from the top half would wither away and not re­gen­er­ate fur­ther, whereas the one from the bot­tom half did re­grow even af­ter many such surg­eries. Is the rea­son that the bot­tom part has the nu­cleus? He an­swered the ques­tion by a di­rect ex­per­i­ment, based on the abil­ity of the cut up­per pieces to be grafted onto lower pieces with the nu­cleus. The grafts took and a new plant re­gen­er­ated, cap and all. The two species Häm­mer­ling chose dif­fered in their cap mor­phol­ogy, one, A. crenu­lata, hav­ing a wavy-edged or crenu­lated cap, the other, A. mediter­ranea (now called A. ac­etab­u­lum), a smooth one. The defin­ing ob­ser­va­tion was that the re­gen­er­ated plant had the mor­pho­log­i­cal char­ac­ter­is­tic of the bot­tom part, which was the first con­clu­sive demon­stra­tion that the nu­cleus con­trols de­vel­op­ment. To con­firm this, he trans­planted nu­clei of one species into an­other and got a hy­brid with char­ac­ter­is­tics of both species. Hämmerling's ex­per­i­ment was the fore­run­ner of all sub­se­quent nu­clear trans­plan­ta­tions, which started some 20 years later with John Gur­don's ex­per­i­ment show­ing that a ma­ture frog could be pro­duced by trans­plant­ing a nu­cleus from an in­testi­nal ep­ithe­lial cell into an enu­cle­ated egg. For a video of Hämmerling's ex­per­i­ment, see here.

Fig­ure 3. The cap of A.acetabulum (top left) is a smooth disc, that of A. crenu­lata (top right) is branched. Häm­mer­ling grafted the stalk of one species onto the foot of an­other. The cap that even­tu­ally de­vel­oped on the grafted cell matched the species of the foot rather than that of the stalk. In this ex­am­ple, the cap that grow­ing on the grafted stalk looks like the base species one, A. ac­etab­u­lum Source

Häm­mer­ling dis­cov­ered an­other sur­pris­ing fact: the anu­cle­ated top por­tions of the cut stalk could not only re­gen­er­ate the caps (al­beit, as men­tioned al­ready, only once), but those pieces far­thest re­moved from the nu­cleus re­gen­er­ated the best caps. This sug­gested that the stalk con­tained sub­stances in­volved in cap mor­pho­gen­e­sis that were dis­trib­uted along a gra­di­ent, the low­est con­cen­tra­tion be­ing at the basal end near the nu­cleus and in­creas­ing to­ward the top of the stalk. He also found that such sub­stances were in­volved in the for­ma­tion of all the parts of the or­gan­ism, in­clud­ing whorls of hairs to­wards the top of the stalk and the rhi­zoid. In later ex­per­i­ments in­volv­ing in­ter­spe­cific grafts, Häm­mer­ling and col­leagues showed that the nu­cleus is the source of these mor­pho­genetic sub­stances.

How Did Ac­etab­u­lar­ias Un­ravel the Cen­tral Dogma?   In his days, Häm­mer­ling could not guess the mor­pho­genetic sub­stances were mes­sen­ger RNA (which was not de­scribed un­til 1961) but he soon de­clared that they "car­ried ge­netic in­for­ma­tion from the nu­cleus to the cy­to­plasm." He thus an­tic­i­pated the dis­cov­ery of mRNA by some 30 years. In time, he and sev­eral other in­ves­ti­ga­tors es­tab­lished that these sub­stances were in­deed mR­NAs. Later on, Jean Bra­chet and col­leagues in Brux­elles stud­ied RNA and pro­tein syn­the­sis in nu­cle­ated and anu­cle­ated Ac­etab­u­laria and showed that RNA flows from the nu­cleus to the cy­to­plasm where pro­teins are syn­the­sized and ul­ti­mately ac­count for the dif­fer­en­ti­a­tion of um­brel­las. (In the 1950's, Brachet's lab was one of the Eu­ro­pean mec­cas for Amer­i­cans to do a post­doc).

What is the cur­rent sta­tus of Ac­etab­u­laria re­search?   Work with this model or­gan­ism has been helped greatly by ma­nip­u­la­tions that shorten its life cy­cle. The life cy­cle used to take some six months in the lab and one to two years in the wild. A se­ries of im­prove­ments in the medium and the use of ax­enic zy­gotes plus other tricks short­ened the life cy­cle to about 97 days, a big jump, con­sid­er­ing. This is not E. coli, but go­ing from zy­gote to zy­gote in that time is not bad com­pared with "higher" plants and an­i­mals.

Al­though its afi­ciona­dos may well ar­gue that Ac­etab­u­laria has not re­ceived its proper share of at­ten­tion of late, con­sid­er­able work has been car­ried out with it in re­cent times. For a re­view, see here. As you would ex­pect, its gi­ant size opened the door to study­ing de­tails of how its con­stituents are dis­trib­uted along its var­i­ous re­gions. For ex­am­ple, it has been shown that spe­cific mR­NAs are dis­trib­uted along the stalk and that they fall into four classes of lo­cal­iza­tion: through­out the or­gan­ism, at the base, at the apex of the stalk, and in lo­ca­tions that change dur­ing de­vel­op­ment. These mol­e­cules are likely trans­ported via actin mi­cro­fil­a­ments of the cy­toskele­ton that span the whole cell. The Ac­etab­u­lar­ias' cy­toskele­ton con­sists of par­al­lel bun­dles of actin that run ax­i­ally and are in­volved in force­ful cy­to­plas­mic stream­ing. Also well stud­ied is the be­hav­ior of the var­i­ous classes of mR­NAs in­volved in the syn­the­sis of struc­tural and reg­u­la­tory pro­teins. It is note­wor­thy that some, no­tably the mR­NAs for tubu­lin, are made and stored long be­fore be­ing used. How is this done? Good ques­tion.

Much in­for­ma­tion has be­come avail­able re­gard­ing cy­toskele­tal and en­domem­brane dy­nam­ics, elec­tro­phys­i­o­log­i­cal el­e­ments af­fect­ing ion fluxes, and syn­the­sis and me­chan­i­cal prop­er­ties of the cell wall. Like else­where, sig­nal trans­duc­tion and hor­monal con­trol are in­volved in reg­u­la­tory path­ways. In ad­di­tion, the ef­fects of en­vi­ron­men­tal fac­tors in­clud­ing light and grav­ity have been stud­ied ex­ten­sively and found to be in­volved in reg­u­la­tion of mor­pho­gen­e­sis.

Open for more work with this or­gan­ism are such fun­da­men­tal ques­tions of de­vel­op­men­tal bi­ol­ogy as how struc­ture and func­tion op­er­ate at lo­cal­ized re­gions and how they are es­tab­lished and main­tained. In the words of D. F. Man­doli, one of the prin­ci­pal present day in­ves­ti­ga­tors of this or­gan­ism: "..., to me, the spe­cial ap­peal of A. ac­etab­u­lum lies not just in the abil­ity to ad­dress im­por­tant ques­tions in de­vel­op­men­tal and struc­tural bi­ol­ogy in the con­text of a phys­i­cally large and ar­chi­tec­turally com­plex uni­cell, but in be­ing able to do so with ac­cess to a di­verse and ro­bust toolkit be­cause this means that if one av­enue of at­tack does not work, an­other prob­a­bly will."