Raft­ing Through Time

by Elio

In the placid old days, cell mem­branes were as­sumed to be fairly sim­ple af­fairs: lipid bi­lay­ers with some pro­teins float­ing in them like corks. This "fluid mo­saic" arrange­ment took a beat­ing around the 1970's, when bio­physi­cists sug­gested that mem­branes may be com­posed of mi­crodomains, where cer­tain lipids and pro­teins be­come seg­re­gated into what be­came known as lipid rafts. With fur­ther work, these turned out to be places where spe­cific lipids such as steroids and sphin­golipids con­cen­trate. The size of the rafts is sup­pos­edly in the 10–200 nm range. Lipid rafts are said to be in­volved in sig­nal trans­duc­tion, pro­tein traf­fick­ing, and more. Some viruses, e.g. in­fluenza, are as­sem­bled at lipid rafts, and the rafts play a role in prion de­vel­op­ment and trans­mis­sion. The sub­ject is not with­out con­tro­versy but it has many ad­her­ents.

Source

Now, what ev­i­dence is there for bac­te­r­ial lipid rafts? In eu­kary­otic cells, mem­branes can be di­vided into de­ter­gent-re­sis­tant (DRM) and de­ter­gent-sen­si­tive (DSM) frac­tions. Al­though the DRM frac­tion may not be the same thing as lipid rafts, it does in­clude pro­teins thought to be present in rafts. Sure enough, when B. sub­tilis mem­branes were treated with de­ter­gent, FloT was found in the DRM frac­tion. In­ci­den­tally, other pro­teins found in this frac­tion are in­volved in sig­nal­ing, mol­e­cule traf­fick­ing, and pro­tein se­cre­tion. Food for thought.

Lipid raft or­ga­ni­za­tion in eu­kary­otic cells. Re­gion (1) is stan­dard lipid bi­layer, while re­gion (2) is a lipid raft. (3) and (4) are mem­brane pro­teins. Source

The au­thors came by the bac­te­r­ial lipid rafts idea in a round­about but in­ter­est­ing way, hav­ing to do with the ef­fects of the sporu­la­tion-re­lated his­ti­dine ki­nase KinC on biofilm for­ma­tion. They found that KinC ac­tiv­ity de­pended on a pro­tein pre­dicted to syn­the­size squa­lene, a pre­cur­sor in the syn­the­sis of sterol and carotenoid lipids. And, as per their ex­pec­ta­tion, KinC was also found in the DRM frac­tion. Un­der the mi­cro­scope, FloT-YFP fu­sion was lo­cated to about six foci along the cy­to­plas­mic mem­brane, and was ob­served to move rapidly. Sure enough, KinC, known pre­vi­ously to lo­cal­ize in foci, co-lo­cal­izes with FloT. And an­other pro­tein, YqfA, that by bioin­for­mat­ics ap­pears to have a char­ac­ter­is­tic flotillin do­main, does too. Does this mean that FloT and YqfA might have some ef­fect on KinC func­tion? In­deed, a dou­ble mu­tant lack­ing both FlotT and YqfA shows de­creased KinC ac­tiv­ity.
 

Flotillin and KinC lo­cal­iza­tion in B. sub­tilis. (A) Cel­lu­lar lo­cal­iza­tion of FloT-YFP (false-col­ored red). (B) Colo­cal­iza­tion of FloT and KinC in a dou­ble-la­beled strain ex­press­ing the trans­la­tional fu­sions FloT-YFP (false-col­ored red) and KinC-CFP (false-col­ored green). Re­gions where the two sig­nals over­lapped ap­pear yel­low in the merge panel. Source

Since other bac­te­ria have flotillin-like pro­teins, the au­thors asked if their find­ings were rel­e­vant to Staphy­lo­coc­cus au­reus and Es­cherichia coli. In staph, a flu­o­res­cent pro­tein fused to the flotillin ho­molog was lo­cated in a sin­gle fo­cus (one raft per cell?), but in E. coli there were more. Some of the pro­teins found in the DRM frac­tion are in­volved in biofilm for­ma­tion. What may one spec­u­late about the func­tion of lipid rafts in bac­te­ria? Lopez and Kolter pre­sume that the com­part­men­tal­iza­tion of spe­cific pro­teins in tightly packed mem­brane ar­eas might fa­cil­i­tate their ac­tiv­ity. Sounds right.

Now, a bit of nos­tal­gia. A long time ago, in 1972 (pre­his­toric times), a grad­u­ate stu­dent, Betsy Green, and I pub­lished a pa­per pur­port­ing to show that the bac­te­r­ial mem­brane is di­vis­i­ble into some 250 bits. How did we ar­rive at such an im­pu­dent con­clu­sion? Betsy grew sev­eral species of bac­te­ria in tri­tium-la­beled glyc­erol, a spe­cific pre­cur­sor of lipids, then "chased" the la­bel by grow­ing the cul­tures in un­la­beled me­dia. Many gen­er­a­tions later, all the la­bel was still re­tained in phos­pho­lipids. At in­ter­vals she took sam­ples for ra­dioau­to­g­ra­phy, which al­lowed her to es­ti­mate the per­cent of cells that were still la­beled.

Cells were grown for sev­eral gen­er­a­tions in the pre­sence of tri­ti­ated glyc­erol, then fil­tered and grown in me­dia with­out the la­bel. Sam­ples taken at dif­fer­ent times were an­a­lyzed by ra­dioau­to­g­ra­phy. The graph shows the pro­por­tion of un­la­beled cells, that is, cells with no pho­to­graphic grain around them. Open cir­cles: E. coli grown in broth. Open stars: Bacil­lus mega­terium grown in rich broth. Open di­a­monds: E. coli grown in min­i­mal medium. Note that all cul­tures be­haved simi­larly. Source

Hard on the eyes, all that mi­cro­scopic count­ing of grains of pho­to­graphic emul­sion. But count she did, and found that all the cells were la­beled un­til some eight gen­er­a­tions had passed. There­after, the pro­por­tion of la­beled cells di­min­ished by a fac­tor of two at each sub­se­quent dou­bling. The in­ter­pre­ta­tion we fa­vored was that the E. coli mem­brane con­sists of 250 "units of con­ser­va­tion" that re­tained their phys­i­cal in­tegrity upon cell growth. Each would av­er­age some 4 × 10⁴ nm². We had no idea what this meant in terms of the chem­i­cal in­di­vid­u­al­ity of each such mem­brane sub­unit. One more rem­i­nis­cence: this pa­per made ab­solutely no im­pact, prob­a­bly be­cause it was pre­ma­ture and had no heuris­tic value at the time. It was never quoted, to my knowl­edge, de­spite the fact that it ap­peared in a high pro­file jour­nal (PNAS). So it goes. At least Betsy got her Ph.D.

Now, does this piece of pa­le­omi­cro­bi­ol­ogy have any­thing to do with the Lopez and Kolter re­sult? At first, this does not seem to be the case be­cause our bits of mem­brane are much smaller than those now re­ported. How­ever, that may not be the telling part of the story. Who knows, maybe the newly found rafts re­tain their in­tegrity over gen­er­a­tions, which is some­thing that could be de­ter­mined.

Ref­er­ence

López D, Kolter R (2010). Func­tional mi­crodomains in bac­te­r­ial mem­branes. Genes & de­vel­op­ment, 24 (17), 1893−1902. PMID 20713508