Go Ahead, Prove that Eu­kary­otic Cells Arose Via En­dosym­bio­sis

by Elio

Say you want to prove once and for all the en­dosym­bi­otic the­ory of the ori­gin of eu­kary­otic cells, sub­stan­tiating that a proto-cell (ac­cord­ing to many, an ar­chaeon) ac­quired a bac­terium that even­tu­ally mor­phed into a mi­to­chon­drion. The best proof re­quires us­ing a time ma­chine to take you back some 1.5 bil­lion years ago to the event, but this tech­nique has proven elu­sive so far. The sec­ond-best thing may be to try to recre­ate stages of the event in the lab­o­ra­tory, some­thing that was in­deed car­ried out re­cently in the labs of Pe­ter Schultz and col­leagues. They re­ported the suc­cess­ful long-term in­tro­duc­tion of a bac­terium into a yeast cell, which sounds like an early step in eu­karyo­ge­n­e­sis. The chimera they con­structed was sta­ble for over 40 gen­er­a­tions, long enough to al­low it to be stud­ied at leisure. How they did it is a good story.

The in­ves­ti­ga­tors started out with yeast cells with de­fec­tive mi­to­chon­dria, pre­sum­ing that since these mu­tants can­not grow on a non-fer­mentable car­bon source such as glyc­erol, they would re­quire the help of an in­tra­cel­lu­lar bac­terium to thrive in this en­vi­ron­ment. Tit for tat, they also en­gi­neered an E. coli to be de­fec­tive and re­quire the host cells' help for sur­vival. The bacterium's de­fi­ciency was in thi­amine (vi­t­a­min B1) syn­the­sis, an es­sen­tial com­po­nent that must be sup­plied by the yeast. Now one de­pends on the other. Since in the glyc­erol medium, the E. coli is the only source of ATP for the yeast, they en­sured that the bac­terium can trans­port ATP, for which they cloned into it the gene for an ADP/ATP translo­case (un­der con­trol of the in­ducible ara pro­moter). For good mea­sure, the E. coli was also en­dowed with GFP, a flu­o­res­cent pro­tein that makes it dis­tinctly vis­i­ble un­der UV light (Fig­ure 1).

Fig­ure 1. Strat­egy to en­gi­neer S. cerevisiae–E. coli en­dosym­biont chimera. (A) Wild-type S. cere­visiae can grow on medium with glu­cose or glyc­erol due to ATP pro­duc­tion by gly­col­y­sis in the cy­to­plasm and ox­ida­tive phos­pho­ry­la­tion in mi­to­chon­dria. (B) Yeast cells with a de­fect in ox­ida­tive phos­pho­ry­la­tion can­not uti­lize glyc­erol for ATP syn­the­sis and can­not grow in the ab­sence of glu­cose. In­tro­duc­tion of E. coli-ex­press­ing ADP/ATP translo­case and SNARE pro­teins into such mu­tant yeast can re­store yeast growth with glyc­erol as the sole car­bon source. Growth of in­tra­cel­lu­lar E. coli is de­pen­dent on thi­amin dipho­sphate (vi­t­a­min B1) pro­vided by yeast. ER, en­do­plas­mic retic­u­lum; G, Golgi ap­pa­ra­tus; M, mi­to­chon­dria; N, nu­cleus; V, vac­uole. Source

Armed with this bac­te­r­ial can­di­date for a mi­to­chon­dr­ial stand-in, how did they in­tro­duce it into the yeast cells? It had been shown be­fore that both bac­te­ria and iso­lated mi­to­chon­dria can be fused to cell wall-less yeast spher­o­plasts in the pres­ence of poly­eth­yl­ene gly­col, a com­pound that in­duces fu­sion be­tween dis­parate cells. The yeast spher­o­plasts ea­gerly ac­cepted the E. coli and, af­ter some fur­ther ma­nip­u­la­tions (to make the host cells en­tirely un­able to make their own ATP), the in­ves­ti­ga­tors were ready to try the nup­tials be­tween yeast and bac­terium. At first, that didn't work very well, as only a few yeast colonies with the de­sired prop­er­ties arose.

This was not en­tirely sur­pris­ing, as bac­te­ria that ha­bit­u­ally live in­side eu­kary­otic cells pos­sess spe­cial­ized de­vices to keep from be­ing di­gested by the host lyso­so­mal degrada­tive ma­chin­ery, which in­cludes the so-called SNARE pro­teins. So, they in­tro­duced into the bac­te­ria a sys­tem to dis­arm SNAREs. They cloned SNARE-in­hibit­ing pro­teins from the in­tra­cel­lu­lar pathogen, Chlamy­dia tra­choma­tis, which, sure enough, al­lowed E. coli to sur­vive in the yeast cells. A few more ma­nip­u­la­tions (us­ing a few more SNARE genes and low-level con­cen­tra­tions of the an­tibi­otic car­beni­cillin), and they fi­nally ob­tained a siz­able pro­por­tion of yeast colonies har­bor­ing in­tra­cel­lu­lar E coli. The in­ves­ti­ga­tors con­firmed that these hy­brid cells had the ge­netic make-up ex­pected for both yeast and bac­terium. Still, the yeast grew more slowly than its wild type, which they at­trib­uted to a va­ri­ety of pos­si­ble mo­ti­va­tions. Af­ter all, who ex­pects per­fec­tion af­ter such in­tri­cate ma­nip­u­la­tions?

See­ing is be­liev­ing, so could the re­searchers find E. coli cells within their yeast cells? Yes, the en­gi­neered yeasts flu­o­resced, in­di­cat­ing they were car­ry­ing the bac­te­r­ial GFP. Did the bac­te­ria be­have like proper mi­to­chon­dria, that is, did they shed many of their genes and be left with a mi­to­chon­dria-like re­duced genome? So far, what has bene shown is the loss of few genes only, but the in­ves­ti­ga­tors ex­pressed the hope many more could be elim­i­nated, their es­sen­tial func­tions be­ing taken over by the yeast. More work and, who knows, this in­tra­cel­lu­lar E. coli may be re­duced to a proper mi­to­chon­drion. Any­how, did the bac­te­ria take on a mi­to­chon­dria-ish shape, thin­ner and more di­verse-look­ing than the bac­te­ria? Not re­ally at least not yet (Fig­ure 2). They still look like the free-liv­ing bac­te­ria, at least un­der a novel tech­nique called Soft-X-ray To­mog­ra­phy (see movie be­low, or down­load movie). I con­fess, I was not ac­quainted with this amaz­ing-sound­ing tech­nique, which is said to be es­pe­cially suited for the imag­ing of in­tact cells at high res­o­lu­tion.

Fig­ure 2. Ul­tra­struc­tural fea­tures of S. cere­visiae–E. coli ΔnadA chimera cells. Seg­mented re­con­struc­tion of a S. cere­visiae–E. coli ΔnadA cell viewed from three dif­fer­ent per­spec­tives (sec­tion planes 1–3). Or­thoslices (Left) at po­si­tions in­di­cated by the black dashed lines in a re­con­structed cell (Right) are shown for each per­spec­tive. The same or­thoslices are over­laid in the Cen­ter col­umn with out­lines in­di­cat­ing seg­mented or­ganelle as­sign­ment. The gray val­ues were gen­er­ated us­ing LACs, with black cor­re­spond­ing to the high­est LAC value. Or­ganelle color key: green, vac­uole; blue, nu­cleus; salmon, mi­to­chon­dria; and yel­low, high LAC value, bac­te­ria-like struc­ture that re­mained unas­signed af­ter seg­men­ta­tion. Source

To re­turn to the point, namely the ori­gin of early eu­kary­otic cells, the au­thors con­clude: "...fea­tures of the par­a­sitic bac­terium en­ergy me­tab­o­lism (e.g., gen­er­a­tion of ATP through TCA and oxy­gen-de­pen­dent oxi­dative phos­pho­ry­la­tion) to­gether with its ADP/ATP translo­case could al­low ex­pan­sion of such col­o­nized ar­chaeal cells into new eco­log­i­cal niches na­tive to the en­dosym­biont (e.g., con­tain­ing oxy­gen and TCA sub­strate mol­e­cules), but pro­hib­i­tive to non­par­a­sitized host cells. In the new niche, the ta­bles are turned: the ar­chaeal host cell be­comes the par­a­site (can­not pro­duce its own ATP) and the en­dosym­bi­otic bac­terium be­comes the prey as the di­rec­tion of the ATP flow is re­versed (just like in our ex­per­i­men­tal sys­tem where yeast is the par­a­site). Such eco­log­i­cal ex­pan­sion of ar­chaea that har­bored a par­a­sitic en­dosym­biont ex­pres­sing an ADP/ATP translo­case could be the first step in evo­lu­tion­ary tran­si­tion from pre­mi­to­chon­dr­ial en­dosym­biont to pro­tomi­to­chon­drion."

As the au­thors rea­son­ably pro­pose, their study opens the door to fur­ther in­ves­ti­ga­tions of the steps that may be plau­si­bly have been in­volved in the evo­lu­tion of mi­to­chon­dria and there­fore of eu­kary­otic cells. Next to hav­ing been there, this is as good as it gets.

Movie for Soft-X-ray To­mog­ra­phy

Leg­end to movie. An­i­mated se­quence show­ing the cor­re­la­tion be­tween LAC LAC (lin­ear ab­sorp­tion co­ef­fi­cient) val­ues in the to­mo­graphic re­con­struc­tion of a S. cere­visiae cox2-60‒E. coli ΔnadA chimera cell and seg­mented struc­tures. Or­thoslices from the re­con­structed cell were sam­pled at reg­u­lar in­ter­vals through­out the vol­ume and dis­played in grey scale (dark is higher LAC value). The seg­mented or­ganelle vol­umes were over­laid into this se­quence of or­thoslices and then shown in iso­la­tion. Color key: blue nu­cleus; salmon mi­to­chon­dria; green vac­uole; grey lipid; yel­low den­sity not at­trib­ut­able to any other ma­jor or­ganelle. Source