VES

OPINION

PERSPECTI
distribution o& lipids in a bila%er can also a&&ect "e"brane cur$ature' Scaffolding proteins and membrane bending. A protein or protein co"ple( can &or" a sca&&old that bends the "e"brane o& an organelle' A growing nu"ber o& proteins that cur$e "e"branes b% the sca&&old "echanis" during $esicular &ission or &usion ha$e been described *&or recent re$iews, see RE+S ,,-.' These proteins bind to "e"branes transientl% and oligo"eri)e, &or"ing rigid, cur$ed struc# tures that bend the underl%ing "e"brane' The oligo"ers disasse"ble when the% are no longer needed' !ore per"anent protein sca&&olds around organelles "ight help to deter"ine their shape' These could be gener# ated b% integral "e"brane proteins, such as ca$eolin, which oligo"eri)es, perhaps into It has long been clear that a co"ple( interpla% o& &actors deter"ines organelle "orpholog%, but how this occurs, and the identit% o& the proteins responsible, has re"ained un2nown' Se$eral recent studies ha$e begun to identi&% and characteri)e proteins that are re/uired &or shaping so"e organelles' These &indings indicate general "echanis"s o& organelle shaping that "ight be used throughout the cell' :ere we discuss &our o& these "echanis"s' +irst, so"e pro# teins help to shape organelles b% stabili)ing "e"brane cur$ature' Second, proteins that tether "e"branes, either to the c%tos2eleton or to other "e"branes, can also deter"ine organelle "orpholog%' Third, the regulated &ission and &usion o& "e"branes can a&&ect organelle shape' ;ast, so"e proteins can help to deter"ine organelle shape b% stabi# li)ing di&&erent "orphologies in continuous "e"branes' Stabili)ing "e"brane cur$ature !aintaining tubules, &enestrations, cisternae and other shapes with high cur$ature re/uires proteins that bend "e"branes' Three t%pes o& "echanis" "ight be used *+I1' 5c.' In the &irst, a protein sca&&old bends the underl%ing "e"brane' In the second, the tendenc% o& the two lea&lets o& a bila%er to re"ain together, 2nown as the bila%er# couple e&&ect, can &acilitate "e"brane bend# ing b% proteins that insert do"ains into one o& the lea&lets' In the third, the as%""etrical

Sheets, ribbons and tubules how organelles get their shape

Gia K. Voeltz Prinz and William A.

Abstract !ost "e"brane#bound organelles ha$e elaborate, d%na"ic shapes and o&ten include regions with distinct "orphologies' These co"ple( structures are relati$el% conser$ed throughout e$olution, which indicates that the% are i"portant &or opti"al organelle &unction' Various "echanis"s o& deter"ining organelle shape ha$e been proposed proteins that stabili)e highl% cur$ed "e"branes, the tethering o& organelles to other cellular co"ponents and the regulation o& "e"brane &ission and &usion "ight all contribute'
Phospholipid bila%ers spontaneousl% &or" spherical or la"inar shaped structures in a/ueous solution' 0hereas so"e organ# elles, such as l%soso"es and pero(iso"es, are relati$el% spherical, "ost ha$e a "ore co"ple( shape' +or e(a"ple, the 1olgi co"ple( and endoplas"ic reticulu" *ER. contain regions that &or" elaborate networ2s o& interconnected cisternae, tubules and &enestrations *+I1' 3.' Other organelles, such as "itochondria and "ulti$esicular bodies, ha$e outer li"it# ing "e"branes and co"ple( networ2s o& internal "e"branes' 1enerating and "aintaining these and other highl% cur$ed organelle "orphologies re/uire speci&ic "echanis"s to stabili)e the"' The shape o& "ost organelles is highl% conser$ed across species4 si"ilarl% shaped organelles are readil% obser$ed in %east and "a""alian cells *+I1' 3.' Also, di&&er# ent organelles can ha$e subdo"ains that rese"ble each other in shape and architec# ture' One e(a"ple is the tubular networ2s &or"ed in regions o& the ER and inner "itochondrial "e"brane *I!!. *co"pare +I1' 3c with +I1' 3e, and +I1' 5a with +I1' 5b.' These networ2s not onl% loo2 si"ilar, but also ha$e tubules with si"ilar dia"eters in nu"erous cell t%pes *appro(i"atel% 67 n" and 87 n" in dia"eter &or ER and I!! tubules, respecti$el%39

4 +I1' 5a,b.' This con# ser$ation indicates that these dia"eters are probabl% i"portant &or organelle &unction'
8

spirals, at the plas"a "e"brane, resulting in locali)ed sites o& "e"brane bending 2nown as ca$eolae *&or a re$iew, see RE+' 6.' An integral "e"brane#protein sca&&old "ight "aintain the high cur$ature and shape o& the tubular cristae o& the I!!' The +3+7#ATP

the "e"brane, thereb% dri$ing tubulation<,>'

In support o& this &unction, inter&ering

with the abilit% o& the s%nthase to di"eri)e "ar2edl% alters the "orpholog% o& I!! in %east, con$erting the I!! tubular cristae into onion#li2e structures?935'

necessar% &or proton pu"ping b% the

s%nthase, indicating that the onion#li2e struc# tures that are &ound in di"eri)ation "utants are not &or"ed as an indirect result o& de&ects in s%nthase &unction' The &or"ation o& I!! tubules is also li2el% to be caused b% oligo# "eri)ation o& the +3+7#

s%nthase is a integral conser$ed and highl% abundant "e"brane#protein

co"ple( in the I!! that has been proposed to a&&ect cristae structure b% two "echanis"s' The di"eri)ation o& the "e"brane# i"bedded +7 portions o&

the +3+7#ATP# s%nthase co"ple( "ight bend the I!!, perhaps b% causing local de&or"ations in

The protein subunits that are re/uired &or di"eri)ation are not

ATP#s%nthase di"ers into chains that "ight &unction as sca&&olds4

electron "icroscop% *E!. re$eals that these oligo"ers spiral around the I!! tubules and there&ore the% "ight &unction as a rigid "e"brane sca&&old that shapes the bila%er<,38 *+I1'

5a.' The dia"eter o& the I!! tubules

PERSPECTIVE
a b

PERSPECTIVE
"ight be deter"ined b% the rise and pitch o& the spirals &or"ed during the oligo"eri)ation o& the +3+7#ATP#s%nthase di"ers'
Bilayer-couple effect. So"e proteins "ight
! 1

! 1 V N 1 ;

2eep the" coupled together, a signi&icant change in the sur&ace area o& either lea&let, N perhaps b% as "uch as a &ew percent-, causes the "e"brane to bend' Proteins can there# &ore cur$e "e"branes b% inserting into onl%
ER

cur$e "e"branes and a&&ect organelle shape' As h%drophobic interactions between the two lea&lets o& a "e"brane bila%er tend to

use the so#called bila%er#couple e&&ect to

c
ER

d
ER tubule

one *or pri"aril% one. lea&let o& a bila%er' +or an integral "e"brane protein to induce or stabili)e "e"brane cur$ature throughout an organelle, it "ust be abun# dant enough to enco"pass the entire region o& the organelle that is being acti$el% shaped' recentl% &or the reticulons and @P3ABop3, which are abundant integral "e"brane proteins that are re/uired to "aintain proper ER tubular structure3,' These

Such a "echanis" has been proposed

;u"en

NE NE

proteins ha$e an unusual "e"brane topolog%4 the% insert two pairs o& C#helices partiall% into the ER "e"brane' The h%drophobic stretches that &or" the hairpin helices are not long enough
to co"pletel% span the "e"brane *the% are onl% 8798, a"ino acids long.' There&ore, these short hairpins probabl% occup% "ore space in the outer than the inner lea&let o& the ER "e"brane, causing it to cur$e and tubulate3, *+I1' 5c.' Interestingl%, these proteins ha$e a si"ilar topolog% to ca$eolin, which also inserts an unusuall% short hairpin into the "e"brane bila%er at the plas"a "e"brane, and this &eature is at least partiall% responsible &or generating "e"brane cur$ature at ca$eolae' Ca$eolin, the +3+7#

e
ER

1olgi stac2

cis &ingers

S ! trans cisternae trans ER

ATP s%nthase, reticulons and @P3ABop3 there&ore share se$eral

proposed "echanistic &eaturesD the% are all oligo"eri)ing integral "e"brane proteins, the% are concentrated at the region that the%

+igure 3 Organelles have comple ! conserved shapes. Electron "icroscop% *E!. i"ages o& the %east Pichia pastoris *a. and a "a""alian nor"al rat 2idne% *NRE. cell *b. showingD the 1olgi stac2 *1., nucleus *N., peripheral endoplas"ic reticulu" *ER., "itochondrion *!. and $acuole or l%soso"e *V or ;.' Note the si"ilar organelle shapes in both cells' Scale bar in a represents 7'- F" and in b represents 3 F"' A con&ocal &luorescence i"age o& a COS cell e(pressing an ER#locali)ed protein labelled with green &luorescent protein *c.' Goth the nuclear en$elope *NE. and the ER are &or"ed &ro" a single, continuous "e"brane' Scale bar represents - F"' E! i"age o& an NRE cell highlights the e$en spacing *H-7 n". o& the two "e"branes o& the NE *d.4 both are continuous with the tubular branches o& the peripheral ER' Scale bar represents 7'5 F"' Single section o& a three# di"ensional to"ogra" o& a chic2 dendrite "itochondrion *e.' The bo(ed sections highlight the conser$ed H57#n" spacing between the inner and outer "itochondrial "e"branes *S. and the H87#

n" dia"eter o& cristae tubules *d.' Scale bar represents 7'3 F"' E! i"age o& an NRE cell showing the "ultiple cisternae o& a 1olgi stac2 *f.' Note the regular intercisternal spacing *H57 n". and the irregular intracisternal lu"enal spacing' Scale bar represents 7'5 F"' Part a is reproduced with per"ission &ro" RE+' -, I *3???. Roc2e&eller =ni$ersit% Press' Part e is reproduced with per"ission &ro" RE+' 3 I *5777. Else$ier' Part c was 2indl%

pro$ided b% J' Rist, Ca"bridge =ni$ersit%, =E' Parts b, d and f were 2indl% pro$ided b% !' ;adins2%, =ni$ersit% o& Colorado, =SA'

PERSPECTIVE

are shaping, and the% ha$e unusual trans"e"brane#do"ain properties that "ight generate "e"brane cur$ature' Tubule &or"ation probabl% re/uires "ore than an increase in "e"brane cur$ature, and the abundant and oligo"eri)ing reticulons and @P3ABop3 probabl% also use a sca&&old# ing "echanis" to stabili)e tubules *this is analogous to ca$eolin at ca$eolae and

PERSPECTIVE

to the +3+7#ATP#s%nthase

co"ple(

in "itochon# drial tubules.' The "ost logical arrange"ent

&or all these proteins would be as spirals or rings that enco"pass the region o& the "e"# brane that the% shape' These structures ha$e not all been decisi$el% de"onstrated b% E!'

ipid asymmetry' Organelle "orpholog%

"ight also be deter"ined b% lipids, so"e o& which cause negati$e or positi$e cur$ature in "e"branes bila%ers when the% are as%""et# ricall% distributed between the two lea&lets' The abilit% o& a lipid to a&&ect bila%er cur$ature is deter"ined b% its e&&ecti$e shape in the "e"brane4 that is, the relati$e cross# sectional area o& its polar head group co"pared with that o& its apolar tail,'

0hen these are si"ilar, &or e(a"ple, in phosphatid%lcholine, the

lipid does not signi&icantl% a&&ect "e"brane cur$ature when it is enriched in one lea&let o& a bila%er' :owe$er, when the relati$e areas o& the head group and tail o& a lipid di&&er, this can pro"ote negati$e or positi$e c cur$ature when as%""etricall% distributed in a "e"brane' E(a"ples o& such lipids include phosphatid%lethana"ine, phosphatidic acid and l%sophosphatid%lcholine' The signi&icant "e"brane de&or"ations that occur during $esicle &or"ation and "e"brane &usion re/uire that such cur$ature#pro"oting lipids beco"e transientl% enriched in s"all regions o& one lea&let o& a "e"brane bila%er,,-,3-'
;u"en Sca&&old

Gila%er#couple e&&ect

;u"en
;ipid ass%"etr%

Indeed, it has been proposed that so"e "e"# brane bending is dri$en b% lipid as%""etries that are generated b% lipid#"odi&%ing
en)%"es or b% &lippases, en)%"es that trans&er lipids between bila%er lea&lets' +or e(a"ple, phospholipase @, which h%drol%ses phos# phatid%lcholine to produce the cur$ature# pro"oting lipid phosphatidic acid, has been i"plicated in se$eral "e"brane# &usion reactions, although its &unction re"ains obscure3693>' The as%""etrical distribution o& lipids that pro"ote "e"brane cur$ature "ight si"ilarl% help to deter"ine organelle shape *+I1' 5c.' :owe$er, unli2e $esicle budding and &usion, the "e"brane de&or"ations re/uired &or "aintaining organelle structures are not transient and "ust occur o$er entire do"ains o& an organelle' There is little e$idence that cur$ature#pro"oting lipids are signi&icantl% and continuousl% as%""etricall% distributed in "ost organelle "e"branes other than the plas"a "e"brane' So"e organelles with elaborate shapes, including the ER and the cis#1olgi co"ple(, are thought to ha$e energ%#independent &lippases that rapidl%

+igure 5 Ma"ing high#curvature membranes. a Electron "icroscop% i"age o& a !eurospora crassa "itochondrion showing the tubular#structure o& the cristae o& the inner "itochondrial "e"brane *I!!.' Tubules ha$e a relati$el% constant dia"eter *H87 n"., intersect at three# wa% Kunctions *indi# cated b% arrows. and ha$e a lu"en that is continuous with the intra"e"brane space *see red bo(.' Scale bar represents 377 n"' b A con&ocal &luorescence "icroscop% i"age o& a COS cell e(pressing green &luorescent protein &used to the hu"an reticulon RTN,a labels the peripheral endoplas"ic reticulu" *ER. networ2 o& tubules' Tubules ha$e a relati$el% constant

dia"eter *-79377 n". and intersect at three#wa% Kunctions *indicated b% the arrow.' Scale bar represents 577 n"' c The high cur$ature o& organelle "e"branes can be generated b% three "echanis"s' In the &irst, an integral "e"brane protein with its trans"e"brane do"ains inserted into the "e"brane bila%er oligo"eri)es to &or" a protein sca&&old with an angle o& cur$ature' The surrounding "e"brane bila%er con&or"s to the shape o& the rigid protein sca&&old' In this e(a"ple, the sca&&old is &or"ed &ro" di"ers o& the + + #ATP s%nthase *shown in red, orange and %ellow.' These can &or" higher order oligo"ers with spiral structures that "ight tubulate the I!! *right.' In the second, a protein "ight occup% a larger area in the outer lea&let o& a "e"brane than in the inner lea&let, causing it to cur$e because o& the bila%er#couple e&&ect, a pheno"enon in which h%drophobic interactions between the two lea&lets o& a "e"brane bila%er tend to 2eep the" coupled together' Reticulon proteins "ight generate cur$a# ture in ER tubules b% this "echanis"' In the third, the as%""etrical distribution o& lipids between the lea&lets o& a "e"brane bila%er can generate cur$ature' +or e(a"ple, "ore or larger phospholipids in the outer lea&let increase the sur&ace area relati$e to that o& the inner lea&let and introduce cur$ature into the "e"brane' Part a is "odi&ied with per"ission &ro" RE+' 5 I *5777. Else$ier' Part b was 2indl% 3 pro$ided b% J' Rist, Ca"bridge =ni$ersit%, =E'

e/uilibrate "ost, i& not all, classes o& lipid in the "e"brane3?953'

It there&ore see"s unli2el% that lipid as%""etries "a2e a signi&icant contribution to deter"ining the shape o& these, or "ost other, organelles'
!e"brane tethering The attach"ent or tethering o& "e"branes to each other or to the c%tos2eleton can also help to deter"ine organelle shape'

"ytos#eletal tethering. In higher eu2ar%otes, "an% organelles, including the ER, "ito# chondria and the 1olgi co"ple(, bind to the c%tos2eleton' These interactions &acili# tate organelle "o$e"ent and positioning in cells but "ight also help to deter"ine organelle "orphologies, which can beco"e se$erel% altered when "icrotubules or actin &ila"ents are depol%"eri)ed' In so"e cases, proteins that are re/uired &or organelle

binding to the c%tos2eleton ha$e been identi&ied' One e(a"ple is the co"ple( o& d%nein and d%nactin that attaches the 1olgi co"ple( to "icrotubules' @isruption o& this co"ple( causes "ar2ed changes in 1olgi shape and locali)ation4 short 1olgi stac2s &or" near ER#e(it sites55'

A si"ilar phenot%pe is seen when "icrotubules are disrupted58' :owe$er, these changes could be indirect because $esicular tra&&ic2ing to

a Tethering "e"brane to c%tos2eleton

the 1olgi co"ple(, which re/uires d%nein, d%nactin and "icrotubules, is needed to "aintain 1olgi shape5,'

!icrotubule

!itochondrial shape is also a&&ected b% both "icrotubule and actin#&ila"ent disruption' In both cases, these changes also see" to be due
to loss o& tra&&ic2ing, speci&icall% o& the &ission protein d%na"in#related protein#3 *@RP34 2nown as @n"3 in %east.5-,56'

spacing between the sheets o& the outer "itochondrial "e"brane and the I!! is regular *H87 n". and is probabl% deter# "ined, at least in part, b% the rigidit% and shape o& the abundant TI!ATO! *trans# locases o& inner and outer "itochondrial "e"brane. translocation co"ple(, which spans both "e"brane bila%ers88'

b Tethering apposing "e"branes

Actin &ila"ents "ight ha$e a "ore direct role

in "aintaining 1olgi shape4 conditions that cause actin depol%"eri)ation without disrupting $esicular tra&&ic2ing cause 1olgi stac2s to &rag"ent and result in swelling o& cisternae5<' The contribution o& the c%tos2eleton to ER structure also re"ains unclear' !icrotubules o&ten align with the tips o& growing ER tubules and "ight be re/uired to e(tend new tubules &ro" the ER *+I1' 8a.' C%tos2eleton#lin2ing "e"brane protein C;I!P68 *pre$iousl% 2nown as p68. is an integral ER#"e"brane protein that binds "icrotubules, and is currentl% the best can# didate protein &or "ediating this interaction' O$ere(pression o& C;I!P68 that lac2s the "icrotubule#binding do"ain causes the ER "e"branes to retract towards the nuclear en$elope and a reduction in ER tubular

!e"brane tethering "ight also help to deter"ine the structure o& I!! cristae' It has been pro# posed that oligo"eri)ation o& "e"brane bound and soluble &or"s o& the I!!# locali)ed d%na"in#li2e protein optic
atroph% protein#3 *OPA34 2nown as !g"3 in %east. can help to deter"ine cristae shape b% tethering these "e"branes to each other' @epletion o& OPA3 results in a "ar2ed reorgani)ation o& the I!! and the loss o& cristae tubules8,,8-' The organi)ation o& 1olgi stac2s al"ost certainl% re/uires "e"brane tethering' 0hereas the intralu"enal spacing in 1olgi cisternae can $ar% across the stac2 &ro" cis to trans *&ro" 3-987 n" &or cis#1olgi and &ro" ,933 n" &or trans#1olgi in a plant cell86.,

c Tethering apposing "e"branes to each

other and to the c%tos2eleton

the intercisternal spacing in 1olgi stac2s is regular8< *H57 n", see +I1' 3&.'
An integral "e"brane#protein "atri( probabl% generates this distance' Indeed, treat"ent o& isolated 1olgi stac2s with so"e proteol%tic en)%"es causes unstac2ing o& cisternae8>'

C%toplas"

Actin

ON!

' Although "icrotubules and C;I!P68 "ight contribute to e(tending and "aintaining the distribution o& ER
structures
5>

+urther"ore, E! has

re$ealed
organi)ed rows o& proteins on the sur&ace o& the 1olgi stac2s that is consistent with an integral "e"brane#protein#"atri( struc# ture, although the identit% o& these proteins is not 2nown86' and alterations in organelle shape *this is also true &or the 1olgi co"ple(.' !oreo$er, ER tubules can be &or"ed in $itro in the absence o& "icrotubules8,5?983'

;INC NE

PNS H-7 n" SS

IN! ;a"ins

tubules throughout the c%toplas", it see"s unli2el% that the% "aintain ER shape4 a&ter ER tubules &or", the% rarel% align with "icrotubules and there is a lag o& "an% "in# utes between "icrotubule depol%"eri)ation
shown' This co"prises the S=N *Sad3 and =NC#>, ho"olog% do"ain. proteins *S., which accu"ulate in the inner nuclear "e"brane *IN!. owing to interactions with the nuclear la"ina' S=N proteins bridge the perinuclear space *PNS. and bind nesprins *N., which are located in the outer nuclear "e"brane *ON!.' The S=N9nesprin co"ple( helps to deter# "ine the highl% conser$ed spacing between the IN! and the ON! *H-7 n".' The nesprins are also anchored to actin surrounding the NE'

Nucleoplas"

+igure 8 Membrane tethering generates organelle shape b$ three mechanisms. a Tethering the "e"brane to co"ponents o& the c%tos2eleton, such as "icrotubules, can deter# "ine organelle shape' An integral "e"brane protein *red c%linder. can bind to an adaptor protein *green o$al. on "icrotubules to allow c%tos2eleton# dependent rearrange"ents o& organelle shape' b Tethering "e"branes to each other can shape and stac2 "e"branes' Stac2ed cisternae can be generated b% di"eri)ation o& integral "e"brane proteins *red c%linders. that are present in apposing "e"brane bila%ers' c Tethering two "e"branes to each other and to the c%tos2eleton shapes the nuclear en$elope *NE.' The ;INC co"ple( is

Other &actors "ight be the pri"ar% deter"inants o& organelle "orpholog%, and "icrotubules could be re/uired "ostl% &or proper dispersal o& organelles in the cell'
%ntermembrane tethering. Attach"ents between "e"branes can also help to deter"ine organelle shape' In so"e cases, these attach"ents cause "e"branes to &or" closel% apposed sheets *+I1' 8b.' +or e(a"ple,

tethering helps to generate the ER cisternae that are seen in so"e cell t%pes' Snapp and co#wor2ers showed that low#a&&init% inter# actions between c%tosolic do"ains o& ER# "e"brane proteins pro"ote the &or"ation o& stac2ed ER cisternae with a &i(ed distance between "e"branes85' Other e(a"ples o& "e"brane tethering that are re/uired &or proper organelle struc# ture co"e &ro" "itochondria' The lu"enal

&ethering to the cytos#eleton and membranes. !e"branes can be si"ultaneousl% teth# ered to both other "e"branes and the c%tos2eleton *+I1' 8c.' The ;INC co"ple( pro$ides a good e(a"ple o& this' This co"ple(, which spans both "e"branes o& the nuclear en$elope, co"prises S=N *Sad3 and =NC#>, ho"olog% do"ain. proteins in the inner nuclear "e"brane *IN!. and nesprins in the

outer nuclear "e"brane' The N ter"inus o& each S=N protein inter# acts with nuclear la"ins, holding the S=N proteins in the IN!, whereas the C ter"inus binds nesprins in the perinu# clear space *PNS., &or"ing a co"ple( that spans the PNS' On the c%toplas"ic &ace o& the nuclear en$elope the nesprins, in turn, interact with the actin c%tos2eleton' The entire co"ple( deter"ines, at least in part, the shape and spacing o& the inner and outer nuclear "e"branes *the PNS is H-7#n" wide.4 depletion o& the S=N proteins results in the e(pansion o& the PNS8?9,3'

;ess &ission

0ild t%pe

;ess &usion

Ldnm'

Lfzo' @eletion o& ()!'

that "aintains the shape o& these organelles is probabl% regulated' There is so"e e$idence that, &or all three o& these "e"# brane s%ste"s, this &usion re/uires co"po# nents o& a &usion "achiner% that includes p?< *also 2nown as $alosin#containing protein *VCP.. and VCIP38-, as antibod# ies against these proteins can pre$ent the asse"bl% o& tubular ER, 1olgi stac2s and the nuclear en$elope in $itro,69,?'

Presu"abl%
b
1olgi stac2s !edial !edial 1olgi stac2 ribbon !edial !edial

1RASP6-

+usion

1!387

the abilit% o& these proteins to pro"ote &usion is regulated b% phosphor%lation or another post#translational "odi&ication during the cell c%cle' Another wa% in which regulated &usion a&&ects organelle "orpholog% has recentl% been described' The 1olgi co"ple( is o&ten arranged as stac2s o& bioche"icall% distinct cisternae' So"e cell t%pes ha$e se$eral o& these stac2s *+I1' 3a. and in "ost $ertebrate cells the stac2s are lin2ed together to &or" ribbons' This process re/uires &usion o& lateral cisternae while still "aintaining the integrit% o& the stac2s *that is, "a2ing sure that all the stac2s do not &use together.' Puthen$eedu et al. ha$e &ound that a co"# ple( o& proteins including 1olgi reasse"bl% stac2ing protein o& 6- 2@ *1RASP6-. and the cis#1olgi "atri( protein o& 387 2@ *1!387. tether lateral cisternae together, thereb% pro"oting their &usion-7 *+I1'

cis

cis

cis

cis

+igure , %alancing fission and fusion affects organelle shape. a regulated

!itochondrial shape is

,b.' The proper shape o& the 1olgi stac2 is

there&ore "aintained b% pro"oting &usion at speci&ic regions o& cisternae' @i&&erentl% shaped do"ains !an% organelles not onl% ha$e co"ple( "orphologies, but also "aintain do"ains in the organelle with di&&erent shapes' Perhaps the "ost stri2ing e(a"ple is the peripheral ER, which is &or"ed &ro" a single "e"brane and is continuous with the nuclear en$elope, but can ha$e do"ains that are tubulated, &enestrated or &or" stac2ed cisternae-3,-5 *+I1'

b% &ission and &usion' Each panel shows "itochondria, labelled with green &luorescent protein, in a single Saccharomyces cere$isiae cell' In wild#t%pe cells, "itochondria &or" branched networ2s o& tubules *"iddle panel.' Cells that lac2 a &usion#pro"oting protein &u))% onions#3 *Lfzo'. contain nu"erous s"all "itochondria *right panel.' @eletion o& the gene &or another &usion protein, "ito# &usin#3 *!+N3., generates a si"ilar phenot%pe' Cells that lac2 a protein needed &or &ission, d%na"in# related protein#3 *Ldnm'., &or" large &enestrated "itochondria *le&t panel.' A sche"atic o& the "itochondrial phenot%pes is shown below' b The continuit% o& the 1olgi#stac2 ribbon is pro"oted b% a &usion "achiner% associated with the oligo"eri)ation o& 1olgi "atri( protein o& 387 2@ *1!3874 red rectangles. and 1olgi reasse"bl% stac2ing protein o& 6- 2@ *1RASP6-, green triangles.' These proteins pro"ote &usion between analogous cisternae to laterall% lin2 adKacent 1olgi stac2s' Cisternae &ro" the cis#1olgi *%ellow. and "edial#1olgi *green. are shown' Con$ersel%, altering the a"ounts o& these proteins results in &rag"entation o& the 1olgi ribbon into indi$idual stac2s' Part a is "odi&ied with per"ission &ro" RE+' ,- I *5773. Else$ier.

3c,d.' :ow di&&er# entl% shaped regions are "aintained in a single "e"brane is not well understood,
o& a &ew large, &enestrated "itochondria,5,,8 *+I1'

Regulating &ission and &usion Organelle "e"branes are d%na"ic and constantl% undergo both &usion and &ission reactions' Not surprisingl%, these processes can pro&oundl% a&&ect organelle shape' A particularl% telling e(a"ple is pro$ided b% "itochondria' !itochondria continu# all% &use with each other and di$ide' 0hen &usion is bloc2ed, b% depleting the proteins

"ito&usin#3 *!+N3. or its %east ho"ologue, 2nown as &u))% onions#3 *+)o3., or OPA3A !g"3, nu"erous s"all "itochondria accu"ulate' G% contrast, bloc2ing &ission, b% depleting @n"3, causes the &or"ation

,a.' Re"ar2abl%, when both &usion and &ission are bloc2ed in cells that lac2 +)o3 and @n"3, "itochondria loo2 si"ilar to those o& wild#t%pe cells' !itochondrial shape is there&ore "aintained b% balancing the rates o& &usion and &ission,,,,-'

In response to en$iron"ental stress, or during the cell c%cle, the balance o& "e"# brane &usion and &ission can o&ten "odulate organelle "orpholog%' +or e(a"ple, the ER, nuclear en$elope and 1olgi co"ple( can co"pletel% disasse"ble during "itosis and then "ust re&or"' The ho"ot%pic &usion

but at least three "echanis"s are thought to be responsible *+I1' -.' One is a selecti$e di&&usion barrier "echanis", which "ight pre$ent "e"brane#shaping proteins or lipids &ro" "o$ing between di&&erent organelle do"ains' A septin#dependent di&# &usion

barrier in the ER at the nec2 o& bud# ding %east cells has recentl% been described4 this barrier slows di&&usion o& "e"brane proteins between the continuous ER o& "other and daughter cells-8' !e"brane tethering probabl% represents a second "echanis" o& generating di&&erentl% shaped regions o& organelles'

a @i&&usion barrier

Plas"a "e"brane

S ER

b !e"brane tethering to obKect

NE Nuclear la"ina Chro"atin

c Inherent shape segregation

+igure - &hree mechanisms of maintaining differentl$ shaped domains in a membrane. a A septin ring *S. located at the bud nec2 o& a %east cell pre$ents rapid di&&usion o& "e"brane proteins between the endoplas"ic reticulu" *ER. o& "other and daughter cell, despite the continu# it% o& the "e"brane' b The spherical shape o& the nuclear en$elope *NE. "ight be partl% deter"ined b% tethering inner nuclear "e"brane proteins *red rectangles. to the nuclear la"ina and to chro# "atin' Tethering o& the inner nuclear "e"brane to the condensed and roughl% spherical nuclear contents con&ers a spherical shape to the nuclear en$elope and pre$ents tubulation o& the NE b% other shaping &actors *&or e(a"ple, reticulons.' c A "e"brane# shaping protein partitions into one do"ain o& a "e"brane because o& its a&&init% &or "e"branes o& a particular shape' :ere, reticu# lon proteins *red. shape ER tubules and locali)e e(clusi$el% to tubules e$en when o$ere(pressed' The shape o& these proteins "ight "a2e it ener# geticall% "ore &a$ourable &or the" to locali)e to highl% cur$ed "e"branes, such as tubules' Con$ersel%, the shape o& the reticulon "ight also pre$ent it &ro" di&&using into "e"branes o& low cur$ature, such as the sheets o& the NE, and there&ore the shape o& the NE is also "aintained'

<'

>'

?'

@ud2ina, N' V', Sunderhaus, S', Graun, :' P' M Goe2e"a, E' J' Characteri)ation o& di"eric ATP s%nthase and cristae "e"brane ultrastructure &ro" Saccharomyces and Polytomella "itochondria' )*BS ett. '(), 8,5<98,85 *5776.' E$erard#1igot, V' et al. +unctional anal%sis o& subunit e o& the + + #ATP s%nthase o& the %east Saccharomyces cere$isiaeD i"portance o& the N#ter"inal "e"brane anchor region' *u#aryot. "ell *, 8,698-- *577-.' Arselin, 1' et al. The "odulation in subunits e and g a"ounts o& %east ATP s%nthase "odi&ies "itochondrial cristae "orpholog%' +. Biol. "hem. +,-, ,78?59,78?? *577,.'

37' 1a$in, P' @', Prescott, !', ;u&&, S' E' M @e$enish, R' J' Cross#lin2ing ATP s%nthase co"ple(es in $i$o eli"inates "itochondrial cristae' +. "ell Sci. ..,, 5888958,8 *577,.' 33' 1iraud, !' +' et al. Is there a relationship between the supra"olecular organi)ation o& the "itochondrial ATP s%nthase and the &or"ation o& cristaeN Biochim. Biophys. Acta .''', 3<,93>7 *5775.' 35' Pau"ard, P' et al. The ATP s%nthase is in$ol$ed in generating "itochondrial cristae "orpholog%' *(B, +. +., 5539587 *5775.' 38' Allen, R' @', Schroeder, C' C' M +o2, A' E' An in$estigation o& "itochondrial inner "e"branes b% rapid#&ree)e deep#etch techni/ues' +. "ell Biol. .)(, 5588955,7 *3?>?.' 3,' Voelt), 1' E', Prin), 0' A', Shibata, B', Rist, J' !' M Rapoport, T' A' A class o& "e"brane proteins shaping the tubular endoplas"ic reticulu"' "ell .+*, -<89->6 *5776.' 3-' !c!ahon, :' T' M 1allop, J' ;' !e"brane cur$ature and "echanis"s o& d%na"ic cell "e"brane re"odelling' !ature */(, -?79-?6 *577-.'

36'

Choi, S'#B', Jen2ins, 1' !', Chan, @' C', Schiller, J' M +roh"an, !' A' A co""on lipid lin2s !&n#"ediated "itochondrial &usion and SNARE#regulated e(oc%tosis' !ature "ell Biol. (, 35--93565 *5776.' Na2anishi, :' et al. Phospholipase @ and the SNARE

+or e(a"ple, proteins residing in the IN! can be tethered to other nuclear co"po# nents, including la"ins and chro"atin, which probabl% helps to 2eep this "e"# brane a di&&erent shape co"pared with the "ostl% tubular peripheral ER-3' ;ast, "e"brane#shaping proteins the"# sel$es could generate di&&erentl% structured organelle regions b% segregating pri"aril% into part o& the organelle' +or e(a"ple, it "ight be energeticall% un&a$ourable &or proteins that stabili)e cur$ed "e"branes to di&&use out o& the" into less cur$ed regions o& an organelle' Such a "echanis" has been proposed &or the reticulons that are highl% enriched in tubulated portions o& the ER3,'

shapes are gener# ated' !e"brane#bending proteins, such as the reticulons, @P3ABop3 and ca$eolin probabl% ha$e a central role in generating shape in "an% organelles' The "ost i"por# tant challenge &or the &uture re"ains under# standing how these proteins &unction and are regulated' Another intriguing /uestion

3<' is the relationship between organelle shape and opti"al organelle &unction' As "ore organelle# shaping proteins are identi&ied, it will be possible to assess this relation# ship "ore directl% and, in particular, to understand how organelle "orpholog% is regulated both during the cell c%cle and during cellular di&&erentiation'

Gia K. Voeltz is at the -epartment of (olecular. "ellular. and -e$elopmental Biology. /ni$ersity of "olorado. Boulder. "olorado 01213. /SA.

William A. Prinz is at the aboratory of "ell Biochemistry and Biology. !ational %nstitute of -iabetes and -igesti$e and Kidney -iseases. !ational %nstitutes of 4ealth. /nited States -epartment of 4ealth and 4uman Ser$ices. Bethesda. (aryland 51035. /SA. e-mails6 gia.$oeltz7colorado.edu8 9prinz7heli:.nih.go$

Re"ar2abl%, this is true e$en when these proteins are o$ere(pressed' There&ore, the a&&init% o& reticulons &or tubulated "e"# branes "ight ensure that regions o& the ER that are de$oid o& reticulons can assu"e shapes other than tubules, such as sheets o& the peripheral ER and nuclear en$elope'
Concluding re"ar2s 0e are still onl% beginning to understand how co"ple( organelle

doiD37'378>Anr"533? Published online < +ebruar% 577<

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8'

,' -' 6'

3>'

3?'

57'

53'

Sso3p are necessar% &or $esicle &usion during sporulation in %east' +. "ell Sci. ..-, 3,769 3,3- *5776.' :uang, P', Altschuller, B' !', :ou, J' C', Pessin, J' E' M +roh"an, !' A' Insulin#sti"ulated plas"a "e"brane &usion o& 1lut, glucose transporter# containing $esicles is regulated b% phospholipase @3' (ol. Biol. "ell .0, 563,95658 *577-.' Gishop, 0' R' M Gell, R' !' Asse"bl% o& the endoplas"ic reticulu" phospholipid bila%erD the phosphatid%lcholine transporter' "ell *+, -39 67 *3?>-.' :err"ann, A', Oachows2i, A' M @e$au(, P' +' Protein# "ediated phospholipid translocation in the endoplas"ic reticulu" with a low lipid speci&icit%' Biochemistry +-, 57589575< *3??7.' Guton, P', !orrot, 1', +ell"ann, P' M Seigneuret, !' =ltra&ast gl%cerophospholipid#selecti$e transbila%er "otion "ediated b% a protein in the endoplas"ic

55'

58'

5,'

reticulu" "e"brane' +. Biol. "hem. +,., 66-3966-< *3??6.' Gur2hardt, J' E', Eche$erri, C' J', Nilsson, T' M Vallee, R' G' O$ere(pression o& the d%na"itin *p-7. subunit o& the d%nactin co"ple( disrupts d%nein# dependent "aintenance o& "e"brane organelle distribution' +. "ell Biol. ./-, ,6?9,>, *3??<.' Cole, N' G', Scia2%, N', !arotta, A', Song, J' M ;ippincott#Schwart), J' 1olgi dispersal during "icrotubule disruptionD regeneration o& 1olgi stac2s at peripheral endoplas"ic reticulu" e(it sites' (ol. Biol. "ell ,, 68396-7 *3??6.' Bang, 0' M Storrie, G' Scattered 1olgi ele"ents during "icrotubule

5-'

56'

5<'

5>'

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5?'

@reier, ;' M Rapoport, T' %n $itro &or"ation o& the endoplas"ic reticulu" occurs independentl% o& "icrotubules b% a controlled &usion reaction' +. "ell Biol. .*(, >>89>?> *5777.' 87' Terasa2i, !', Chen, ;' G' M +uKiwara, E' !icrotubules and the endoplas"ic reticulu" are highl% interdependent structures' +. "ell Biol. .)/, 3--<93-6> *3?>6.' 83' Turner, J' R' M Tarta2o&&, A' !' The response o& the 1olgi co"ple( to "icrotubule alterationsD the roles o& "etabolic energ% and "e"brane tra&&ic in 1olgi co"ple( organi)ation' +. "ell Biol. .)-, 57>3957>> *3?>?.' 85' Snapp, E' ;' et al. +or"ation o& stac2ed ER cisternae b% low a&&init% protein interactions' +. "ell Biol. .0/, 5-<956? *5778.' 88' Endo, T', Ba"a"oto, :' M Esa2i, !' +unctional cooperation and separation o& translocators in protein i"port into "itochondria, the double#"e"brane bounded organelles' +. "ell Sci. ..0, 85-?9856< *5778.' 8,' Cipolat, S', !artins de Grito, O', @al Oilio, G' M Scorrano, ;' OPA3 re/uires "ito&usin 3 to pro"ote "itochondrial &usion' Proc. !atl Acad. Sci. /SA .)., 3-?5<93-?85 *577,.' 8-' +re))a, @' et al. OPA3 controls apoptotic cristae re"odeling independentl% &ro" "itochondrial &usion' "ell .+0, 3<<93>? *5776.' 86' Staehelin, ;' A', 1iddings, T' :' J', Eiss, J' O' M Sac2, +' @' !acro"olecular di&&erentiation o& 1olgi stac2s in root tips o& Arabidopsis and !icotiana seedlings as $isuali)ed in high pressure &ro)en and &ree)e# substituted sa"ples' Protoplasma .',, <-9?3 *3??7.' 8<' ;adins2%, !' S', !astronarde, @' N', !cIntosh, J' R', :owell, E' E' M Staehelin, ;' A' 1olgi structure in three di"ensionsD &unctional insights &ro" the nor"al rat 2idne% cell' +. "ell Biol. .**, 338-933,? *3???.'

8>'

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,?'

=chi%a"a, E' et al. VCIP38-, a no$el essential &actor &or p?<A7,<#"ediated "e"brane &usion, is re/uired &or 1olgi and ER asse"bl% in $i$o' +. "ell Biol. .'-, >--9>66 *5775.' -7' Puthen$eedu, !' A', Gachert, C', Puri, S', ;anni, +' M ;instedt, A' @' 1!387 and 1RASP6-#dependent lateral cisternal &usion allows uni&or" 1olgi#en)%"e distribution' !ature "ell Biol. (, 58>95,> *5776.' -3' !attaK, I' 0' Sorting out the nuclear en$elope &ro" the endoplas"ic reticulu"' !ature ;e$. (ol. "ell Biol. ', 6-96? *577,.' -5' Shibata, B', Voelt), 1' E' M Rapoport, T' A' Rough sheets and s"ooth tubules' "ell .+0, ,8-9,8? *5776.' -8' ;uede2e, C' et al. Septin#dependent co"part"entali)ation o& the endoplas"ic reticulu" during %east polari)ed growth' +. "ell Biol. .0-, >?<9?7> *577-.' -,' Rossanese, O' 0' et al. 1olgi structure correlates with transitional endoplas"ic reticulu" organi)ation in Pichia pastoris and Saccharomyces cere$isiae' +. "ell Biol. .*', 6?9>3 *3???.'

Ac"nowledgements
0e than2 !' ;adins2% and J' Rist &or pro$iding i"ages, and B' Shibata and J' :inshaw &or reading the "anuscript' 0'P' was supported b% the Intra"ural Research Progra" o& the National Institute o& @iabetes and @igesti$e and Eidne% @iseases'

Competing interests statement

The authors declare no co"peting &inancial interests'

@ATAGASES
&he following terms in this article are lin"ed online to1 =niProtEGD httpDAAca'e(pas%'orgAsprot @RP3 1!387 1RASP6- !+N3 OPA3 p68 VCIP38VCP Bop3 Access to this interactive lin"s bo is free online.