The Slime That Smiles

by Heidi Ar­jes

Have you ever won­dered how in­di­vid­ual fin­gers form? If you have taken a de­vel­op­men­tal bi­ol­ogy class, you know that the hand first de­vel­ops as a mit­ten-like struc­ture with the fu­ture fin­gers con­nected (Fig­ure 1). Later, dur­ing nor­mal de­vel­op­ment, the cells in the ar­eas be­tween the fin­gers un­dergo pro­grammed cell death, and thus leave be­hind five fin­gers on each hand. Pro­grammed cell death is im­por­tant not only in de­vel­op­ment and long term vi­a­bil­ity of an­i­mals, but also plays a role in uni­cel­lu­lar or­gan­isms. For ex­am­ple, in the so­cial amoeba Dic­tyostelium, a sub­set of cells un­der­goes a type of pro­grammed cell death sim­i­lar to au­tophagy to form fruit­ing body stalks. The re­main­ing dead cells in the stalk are highly vac­uo­lated and pro­vide sup­port for the spore body (fig­ure 2).

Fig­ure 1: Cell death oc­curs be­tween the emerg­ing dig­its dur­ing de­vel­op­ment. A flu­o­res­cent chem­i­cal was used to vi­su­al­ize re­gions of cell death in these em­bry­onic mouse limbs. Source

Pro­grammed cell death has also been ob­served in bac­te­ria. Some ex­am­ples have been sum­ma­rized in this re­view, in­clud­ing the toxin-an­ti­toxin ad­dic­tion sys­tems. Ev­i­dence of bac­te­r­ial pro­grammed cell death sup­ports the the­ory that prokary­otic or­gan­isms pos­sess the ca­pa­bil­ity to in­sure their demise un­der con­di­tions of over­whelm­ing cell stress. While pro­grammed cell death may not ben­e­fit a sin­gle bac­te­r­ial cell, in sev­eral cases, it can be of ut­most im­por­tance to the sur­round­ing bac­te­r­ial pop­u­la­tion. Many so­cial be­hav­iors and de­vel­op­men­tal processes, in­clud­ing pro­grammed cell death, have been char­ac­ter­ized in biofilms, which are or­ga­nized bac­te­r­ial com­mu­ni­ties where cells com­mu­ni­cate via spe­cial sig­nal­ing path­ways and dif­fer­en­ti­ate into sev­eral dis­tinct cell types. For in­stance, pre­vi­ous re­search on biofilms formed by the sporu­lat­ing bac­te­ria Bacil­lus sub­tilis re­vealed that starv­ing cells sig­nal their neigh­bors to un­dergo pro­grammed cell death. Nu­tri­ents re­leased by the dy­ing cells in this strik­ing dis­play of prokary­otic al­tru­ism al­low the re­main­ing cells to tem­porar­ily de­lay sporu­la­tion. These stud­ies sug­gest that prokary­otic sys­tems like the ones above may have evolved into the more elab­o­rate pro­grammed cell death sig­nal­ing cas­cades of eu­kary­otes.

Fig­ure 2: The stalk cells of Dic­tyostelium un­dergo pro­grammed cell death. In panel C, dead cells are la­beled with pro­pid­ium io­dine, which la­bels DNA of cells with com­pro­mised mem­branes. The vac­uoles are un­la­beled. Source

A new re­port ini­tially caught my eye due to the Sci­ence mag­a­zine fea­ture A wrin­kle in slime and all the smi­ley faces that had been ar­ti­fi­cially in­duced in B. sub­tilis biofilms (Fig­ure 3). How were the au­thors able to ac­com­plish this amaz­ing feat of biofilm en­gi­neer­ing? By se­lec­tively la­bel­ing dead and dy­ing cells in biofilms with flu­o­res­cent chem­i­cals, the au­thors show that ar­eas of cell death ul­ti­mately be­come the lo­ca­tion of wrin­kles in biofilms (Fig­ure 4). Cells in biofilms se­crete ex­tra­cel­lu­lar ma­trix com­po­nents such as poly­sac­cha­rides and amy­loid fibers. When cells grow and di­vide, the ad­di­tional mass pushes on the ex­ist­ing ma­trix, gen­er­at­ing an ever in­creas­ing force on cells. Lo­cal­ized ar­eas of cell death al­low the biofilm sur­face to buckle, re­liev­ing the ex­cess me­chan­i­cal force and al­le­vi­at­ing the stress on in­di­vid­ual cells. In sup­port of this model, the au­thors demon­strate that mu­tant biofilms with­out an ex­tra­cel­lu­lar ma­trix ex­hibit uni­form cell death (in­stead of the wild-type lo­cal­ized pock­ets) and form a wrin­kle-free biofilm. Thus, the ex­tra­cel­lu­lar ma­trix is cru­cial to re­strict the ar­eas of cell death. The ex­cess force dis­trib­uted through the ex­tra­cel­lu­lar ma­trix un­der­lies lo­cal­ized cell death in wild-type biofilms. In ad­di­tion, the au­thors show that patches of cell death cor­re­sponded to ar­eas where orig­i­nally there was an in­creased cell den­sity. In a neat demon­stra­tion, they ar­ti­fi­cially place ini­tial ar­eas of high cell den­sity in cool pat­terns such as let­ters and smiles. As the biofilms de­vel­oped, these ar­eas were more likely to un­dergo cell death and wrin­kle into the author's de­signs.

Fig­ure 3: Let­ters and smi­ley faces ar­ti­fi­cially en­gi­neered on B. sub­tilis biofilms. The au­thors har­ness the power of cell death to en­gi­neer wrin­kle de­signs on the biofilm sur­face. Source

This pa­per shows that cell death helps gen­er­ate 3D biofilm wrin­kles by giv­ing the biofilm sur­face room to buckle. I couldn't help but no­tice its par­al­lels with pro­grammed cell death dur­ing de­vel­op­ment. The cells in the biofilm en­counter a me­chan­i­cal stress due to in­creas­ing cell den­sity. Some­how the cells "sense" this in­creased den­sity, per­haps through forces gen­er­ated by the ex­tra­cel­lu­lar ma­trix. These cells die in or­der to re­lieve the ten­sion in the sur­face of the biofilm and help cre­ate wrin­kles. In ad­di­tion to the de­vel­op­men­tal processes of biofilms re­viewed here, this pro­vides an ex­am­ple of the im­por­tance of so­cial in­ter­ac­tions in bac­te­r­ial biofilm de­vel­op­ment. In con­trast to the pro­grammed cell death un­der­taken to de­lay sporu­la­tion, by form­ing wrin­kles, lo­cal­ized death can ben­e­fit the en­tire biofilm in nu­tri­ent rich en­vi­ron­ments as well as nu­tri­ent poor en­vi­ron­ments. No­tably, biofilm wrin­kles have been shown to de­ter the ef­fi­cacy of an­tibi­otics dis­solved in liq­uids or ap­plied by gases, as well as ethanol and other an­ti­sep­tic cleansers. The re­sult­ing in­crease in sur­face area may also help cells ob­tain nu­tri­ents and ex­pel waste.

Fig­ure 4: Wild-type biofilm wrin­kling (A) oc­curs in ar­eas where cell death has pre­vi­ously oc­curred (E and F). Cell death un­der­lies wrin­kle for­ma­tion by pro­mot­ing biofilm buck­ling (C and D). Source

The re­sults of this study bring up many in­ter­est­ing ques­tions. I am cu­ri­ous if the cells that die to en­able wrin­kling ex­hibit the hall­marks of bac­te­r­ial apop­to­sis ob­served in this pa­per. I also won­der about the sig­nals that in­duce these spe­cific cells to die: is pure me­chan­i­cal force the trig­ger or does this process in­volve a sig­nal se­creted from neigh­bor­ing cells, sim­i­lar to the can­ni­bal­is­tic cells that live off nu­tri­ents from dy­ing neigh­bors men­tioned above. An­swer­ing these ques­tions will add new in­sights on how cell death to form biofilm wrin­kles com­pares to known mech­a­nisms of bac­te­r­ial pro­grammed cell death. In ad­di­tion, the author's demon­stra­tion of en­gi­neer­ing biofilm struc­tures can be used in syn­thetic bi­ol­ogy and ma­te­r­ial sci­ences and could pro­pel new cut­ting edge ex­per­i­ments and de­signs in tis­sue en­gi­neer­ing.

Buddy said it best in the movie Elf, "I just like to smile. Smiling's my fa­vorite!" I am sure these bac­te­r­ial emoti­cons have made count­less peo­ple smile and mar­vel at the prokary­otic won­ders that never cease to amaze.

Heidi Ar­jes is a grad­u­ate stu­dent in the Levin Lab in the De­part­ment of Bi­ol­ogy at Wash­ing­ton Uni­ver­sity in St. Louis.