Engineering eukaryotic protein factories The biopharmaceuticals market is currently outperforming the pharmaceuticals market and is now valued at US$ 48 billion with an average annual growth of 19 % . Behind this success is a 100-fold increase in productivities of eukaryotic expression systems . However , the productivity per cell has remained unchanged for more than 10 years . The engineering of the ER-resident protein folding machinery is discussed together with an overview of signal transduction pathways activated by heterologous protein overexpression to increase cell specific productivities . Introduction The demand for therapeutic proteins , i.e. monoclonal antibodies , has been forecast to exceed production capacities -LRB- Werner 2004 -RRB- . Moderate increases in product titers and yields are expected to result in significant savings in capital investment and consumer costs -LRB- Werner 2004 -RRB- . Over the last decade product titers have increased from ∼ 20 mg l − 1 to 2 g l − 1 -LRB- Werner 2004 ; Wurm 2004 -RRB- . These improvements are largely attributable to increases in viable cell densities . However , cell specific productivities remained nearly constant during this time -LRB- Wurm 2004 -RRB- . To improve cell specific productivities the bottleneck for heterologous protein secretion has to be identified and resolved . In expression systems exploiting multiple gene copies or strong promoters the amount of secreted heterologous protein does not increase proportionally with gene copy number -LRB- Schröder 2007 -RRB- , messenger ribonucleic acid -LRB- mRNA -RRB- -LRB- Schröder et al. 1999 -RRB- , or even the intracellular amount of the heterologous protein -LRB- Schröder and Friedl 1997 -RRB- . In these cells , the protein accumulates in intracellular aggregates -LRB- Schröder and Kaufman 2005 -RRB- , associates with the molecular chaperone heavy chain-binding protein -LRB- BiP -RRB- / glucose-regulated protein of 78 kDa -LRB- GRP78 -RRB- / karyogamy 2 protein -LRB- Kar2p -RRB- -LRB- Schröder and Kaufman 2005 -RRB- , and induces dilation of the endoplasmic reticulum -LRB- ER -RRB- -LRB- Dorner et al. 1989 ; Gennaro et al. 1991 -RRB- . Therefore , exit of the correctly folded polypeptide chain from the ER is the rate-limiting step for heterologous protein secretion . I will discuss strategies to improve cell specific productivities by engineering protein folding in the ER and signal transduction pathways activated by overexpression of heterologous proteins . Protein folding in the ER The ER-resident protein folding machinery has four components : -LRB- 1 -RRB- Molecular chaperones assisting other proteins to fold , -LRB- 2 -RRB- enzymes such as protein disulfide isomerases -LRB- PDIs -RRB- and cis-trans peptidyl prolyl isomerases -LRB- PPIs -RRB- , -LRB- 3 -RRB- a degradation machinery , and -LRB- 4 -RRB- signal transduction pathways that co-ordinate protein folding demand with capacity . At least three hierarchically organized chaperone systems operate in the ER , the heat shock protein of 70 kDa -LRB- HSP70 -RRB- chaperones BiP and luminal HSP seventy 1 protein -LRB- Lhs1p -RRB- / chaperone in the ER 1 protein/Ssi1p/GRP170 / oxygen-regulated protein of 150 kDa -LRB- ORP150 -RRB- , the HSP90 chaperone GRP94/adenotin/endoplasmin / tumor rejection antigen glycoprotein of 96 kDa -LRB- gp96 -RRB- / ER protein of 99 kDa/HSP108/Ca2 + - binding protein 4/protein kinase of 80 kDa , and the lectin chaperones calnexin/p88 , calreticulin/Ca2 + - binding protein of 63 kDa -LRB- CAB-63 -RRB- / calregulin/Ca2 + - regulated protein of 55 kDa -LRB- CRP55 -RRB- / high affinity Ca2 + - binding protein -LRB- HACBP -RRB- , and calmegin/calnexin-t -LRB- Fig. 1 -RRB- . BiP works on completely unfolded polypeptide chains and translocates nascent polypeptide chains into the ER , whereas GRP94 and the lectin chaperones work on partially folded substrates . Fig. 1Hierarchy of chaperone systems in the ER . Abbreviations : Glc = d-glucose , Pi = inorganic phosphate -LRB- -RRB- BiP consists of an N-terminal ATPase domain and a C-terminal substrate binding domain . BiP cycles through rounds of adenosine triphosphate -LRB- ATP -RRB- hydrolysis and adenosine diphosphate -LRB- ADP -RRB- ATP exchange -LRB- Fig. 1 -RRB- , which makes BiP a chaperone foldase -LRB- Winter and Jakob 2004 -RRB- . At least six DnaJ or HSP40 co-chaperones stimulate the ATPase activity of BiP and two growth after phage induction E -LRB- GrpE -RRB- co-chaperones the ADP ATP exchange reaction . In the ADP-bound form BiP has high affinity for unfolded substrates . Substrates bound to BiP are conformationally locked . ADP ATP exchange decreases the affinity of BiP for unfolded substrates . Substrate binding stimulates the ATPase activity of BiP . Thus , protein folding requires ATP -LRB- Dorner and Kaufman 1990 -RRB- . BiP and Lhs1p coordinate their activities . Lhs1p is a nucleotide exchange factor for BiP , and BiP stimulates the ATPase activity of Lhs1p -LRB- Steel et al. 2004 -RRB- . Cytosolic HSP90 chaperones are ATP-consuming chaperone foldases -LRB- Fig. 1 -RRB- regulated by several co-chaperones that affect its ATPase activity , load substrates onto HSP90 chaperones , assist in protein folding , and target substrates to degradation pathways -LRB- Strudwick and Schröder 2007 -RRB- . GRP94 co-chaperones have not been identified , but other ER luminal chaperones found in complexes with GRP94 may be GRP94 co-chaperones . The KM value of GRP94 for ATP is ∼ 100 μM and its intrinsic ATPase activity is barely detectable . These data indicate that GRP94 may be a chaperone holdase , a chaperone that binds to unfolded proteins , but does not cycle through ATP hydrolysis cycles . A drop of adenine nucleoside concentrations in stress situations may activate GRP94 -LRB- Rosser et al. 2004 -RRB- . The lectin chaperones share an N-terminal globular oligosaccharide-binding domain and a C-terminal extended hairpin loop , the P domain , which provides the chaperone function -LRB- Strudwick and Schröder 2007 -RRB- . Calnexin and calmegin are transmembrane proteins . Calreticulin is a soluble protein . Most proteins entering the ER are glycosylated by addition of the oligosaccharide Glc3Man9GlcNAc2 -LRB- Glc = d-glucose , Man = d-mannose , GlcNAc = N-Acetyl-2-d-glucosamine -RRB- . α-Glucosidases I and II rapidly remove the two terminal d-glucose residues . Calnexin and calreticulin bind to this monoglucosylated oligosaccharide . Removal of the third d-glucose moiety by α-glucosidase II releases the protein from calnexin/calreticulin . Reglucosylation of unfolded proteins by uridine diphosphate -LRB- UDP -RRB- - glucose : glycoprotein glucosyl transferase -LRB- UGGT -RRB- triggers repeated interactions with calnexin/calreticulin -LRB- Fig. 1 -RRB- . This reglucosylation reaction consumes UDP-glucose . Therefore , the calnexin chaperones are chaperone foldases . The Saccharomyces cerevisiae genome encodes a calnexin homolog , but no homolog for UGGT , suggesting that the lectin chaperones may also function as holdases . Demannosylation of N-linked oligosaccharides trigger export of proteins to the Golgi complex or their targeting for retrotranslocation into the cytosol and proteasomal degradation -LRB- Lederkremer and Glickman 2005 ; Ruddock and Molinari 2006 -RRB- . An expanding family of PDIs catalyzes the formation and isomerization of disulfide bonds . Disulfide bond formation requires the regeneration of oxidized PDI catalyzed by the flavin adenine dinucleotide -LRB- FAD -RRB- - dependent oxidases ER oxidation 1 protein -LRB- Ero1p -RRB- / ERO1-Lα , ERO1-Lβ and essential for respiration and viability 2 protein -LRB- Erv2p -RRB- -LRB- Fig. 2 -RRB- . The final electron acceptor is molecular oxygen . Oxidative protein folding accounts for ∼ 25 % of all reactive oxygen species -LRB- ROS -RRB- formed in a cell -LRB- Tu and Weissman 2004 -RRB- and is increased by ER stress -LRB- Haynes et al. 2004 -RRB- . Disulfide bond isomerization is independent of the Ero1p oxidases . Reduced glutathione provides reducing equivalents to remove incorrectly formed disulfide bonds . PDI has chaperone holdase activity that is independent of its protein disulfide isomerase activity -LRB- Wilkinson and Gilbert 2004 -RRB- and redox-regulated foldase activity -LRB- Tsai et al. 2001 -RRB- . Fig. 2Disulfide bond formation and isomerization reactions catalyzed by PDI Slowly folding proteins or folded incompetent proteins have to be removed from chaperone cycles to prevent them from poisoning the protein folding machinery . This is achieved by targeting these proteins for retrotranslocation into the cytosol and degradation by the proteasome in a process called ER associated protein degradation -LRB- ERAD -RRB- -LRB- Yoshida 2007 -RRB- -LRB- Fig. 3 -RRB- . Proteins destined for ERAD are recognized by BiP , PDI , and lectin sensors such as ER-degradation enhancing α-mannosidase-like proteins 1 -- 3 -LRB- EDEM1 -- 3 -RRB- / homologous to mannosidase I 1 protein -LRB- Htm1p -RRB- / mannosidase-like 1 protein -LRB- Mnl1p -RRB- and osteosarcoma-9 -LRB- OS-9 -RRB- / yeast OS-9 homolog protein -LRB- Yos9p -RRB- -LRB- Gauss et al. 2006a -RRB- . The lectin sensor EDEM extracts unfolded proteins from the calnexin cycle . ERAD substrates are then retrotranslocated into the cytosol via the secretory 61 -LRB- SEC61 -RRB- channel or a channel formed by derlin proteins , ubiquitinated by action of ubiquitin-conjugating enzymes -LRB- E2 enzymes -RRB- and ubiquitin ligases -LRB- E3 enzymes -RRB- , deglycosylated , and degraded by the proteasome . Redundancy exists in the ubiquitination machinery involved in ERAD . In yeast and mammalian cells at least three distinct E2 · E3 complexes are present in the ER membrane . The substrate specificities of these complexes , as well as their substrate selection rules are poorly understood . In yeast , the E3 ligase Hrd1p co-operates with Hrd3p and Yos9p to select its substrates -LRB- Gauss et al. 2006b -RRB- . Another selection criterion is the time an unfolded protein spends in foldase cycles . In the calnexin cycle , demannosylation of N-linked oligosaccharides generates glycostructures recognized by lectins such as EDEM1 -- 3 . Association of cytosolic HSP70s with the nucleotide exchange factor B cell leukemia/lymphoma 2 -LRB- Bcl-2 -RRB- associated athanogene protein 1 -LRB- BAG-1 -RRB- -LRB- Alberti et al. 2003 -RRB- and of cytosolic HSP90s with the ubiquitin ligase carboxyl terminus of heat shock cognate protein of 70 kDa -LRB- HSC70 -RRB- interacting protein -LRB- CHIP -RRB- -LRB- Höhfeld et al. 2001 -RRB- targets proteins for proteasomal degradation . Association of the DnaJ co-chaperone 58 kDa inhibitor of double-stranded RNA activated protein kinase -LRB- PKR , p58IPK -RRB- with HSP70 chaperones at the translocation pore targets proteins , whose translocation into the ER is stalled , for proteasomal degradation -LRB- Oyadomari et al. 2006 -RRB- . If similar targeting mechanisms for proteins folding slowly in the BiP foldase cycle exist , is currently unknown . Fig. 3ER associated protein degradation . The exact nature of the retrotranslocation channel is unknown . The list of E2 and E3 enzymes catalyzing ubiquitination of ERAD substrates is not exhaustive . Abbreviations : NEF = HSP70 co-chaperone , Ub = ubiquitin Engineering of chaperone machineries Engineering of chaperone holdases should be straightforward . Holdases hold onto an unfolded protein and act in an equimolar ratio to the unfolded substrate , but are unable to support folding reactions . Holdases may provide a buffer capacity to prevent aggregation of unfolded proteins . Co-overexpression of chaperone foldases may be beneficial to overexpression of holdases -LRB- Smith et al. 2004 ; Zhang et al. 2006b -RRB- . Overexpression data for calnexin/calreticulin and PDI are most easily explained by assuming that this buffer capacity is increased . Calnexin or calreticulin overexpression generally improved heterologous protein secretion -LRB- Chung et al. 2004 ; Conesa et al. 2002 ; Kato et al. 2005 -RRB- . Overexpression of one cycle constituent should not increase cycle capacity , if this constituent is not limiting , and if constituent levels are not co-regulated . The hypothesis that calnexin or calreticulin are limiting for the calnexin cycle seems unlikely , because of the high abundance of these proteins . Alternatively , overexpression of calnexin or calreticulin may inactivate lectins such as EDEM -LRB- Molinari et al. 2003 ; Oda et al. 2003 -RRB- , which target unfolded proteins for ERAD . Further , elevated calnexin or calreticulin levels not supported by concomitant increases in activities of other calnexin cycle components , may convert these surplus amounts of calnexin and calnexin into holdases or inactive chaperones . PDI overexpression increased secretion of some , but not all , heterologous proteins -LRB- Butz et al. 2003 ; Damasceno et al. 2006 ; Inan et al. 2006 ; Kato et al. 2005 ; Mohan et al. 2007 ; Schröder 2007 ; Zhang et al. 2006b -RRB- . PDI overexpression provides increased holdase activity in the ER . For example , overexpression of catalytically inactive PDI improved heterologous protein secretion -LRB- Hayano et al. 1995 -RRB- . PDI overexpression also improved secretion of heterologous proteins containing no disulfide bonds -LRB- Powers and Robinson 2007 ; Smith et al. 2004 ; Vad et al. 2005 -RRB- . Substrate selectivity of PDI may explain why PDI overexpression failed to increase heterologous protein secretion in some studies -LRB- Butz et al. 2003 ; Damasceno et al. 2006 ; Mohan et al. 2007 ; Schröder 2007 -RRB- . If this is the case , co-expression of an alternative holdase , i.e. GRP94 , may yield more consistent results for different heterologous proteins . Alternatively , increased rates of disulfide bond formation or isomerization in PDI overexpressing cells may explain increased heterologous protein secretion . As an enzyme , PDI should function at substochiometric ratios to its substrates . In disulfide bond formation , regeneration of oxidized PDI may become rate-limiting in PDI overexpressing cells . PDI is an abundant ER protein , making it unlikely that its overexpression can significantly augment its catalytic activities . Engineering of foldases seems more difficult , because of the complex regulation of these chaperones -LRB- see above -RRB- . BiP overexpression increased heterologous protein secretion for about half of the heterologous proteins studied -LRB- Chung et al. 2004 ; Damasceno et al. 2006 ; Dorner et al. 1992 ; Hsu and Betenbaugh 1997 ; Kim et al. 2003 ; Schröder 2007 ; Smith and Robinson 2002 ; Zhang et al. 2006b -RRB- , but can also have negative effects -LRB- Dorner et al. 1992 ; Schröder 2007 -RRB- . In cells overexpressing BiP one of its co-chaperones , Lhs1p , or ER luminal ATP may become limiting , converting BiP into a holdase or inactive chaperone . This may target proteins towards ERAD , stall translocation , convert Lhs1p into a holdase or inhibit Lhs1p . Increased BiP activity may stall the GRP94 and/or calnexin -- calreticulin chaperone machineries , because of the hierarchy of ER luminal chaperone systems . This again may target heterologous proteins for ERAD . BiP overexpression attenuates the unfolded protein response -LRB- UPR , see below -RRB- . Thus , BiP levels in WT and BiP overexpressing cells expressing a heterologous protein may not be dramatically different . Further , BiP overexpressing cells may experience an imbalance in their chaperone machineries , because the UPR co-ordinates expression of several chaperones . Moreover , if overexpression of BiP shifts the folding bottleneck to the GRP94 and calnexin chaperone systems , upregulation of these chaperone systems by the UPR may be blunted , because of suppression of UPR activation by elevated BiP levels . Unfolded protein response -LRB- UPR -RRB- ER stress is the perturbation of the balance between the folding capacity and the folding demand imposed on the ER . To restore ER homeostasis , the UPR activates expression of chaperone , PDI , PPI , and ERAD genes , attenuates transcription of genes encoding secretory proteins and general translation , induces phospholipid synthesis , and induces an inflammatory response . Apoptosis is induced if these actions fail to restore ER homeostasis or in response to prolonged ER stress -LRB- Schröder and Kaufman 2005 -RRB- . The ER membrane of higher eukaryotes harbors at least three classes of transmembrane proteins activated by ER stress , basic leucine zipper -LRB- bZIP -RRB- transcription factors synthesized as type II transmembrane proteins , of which the best characterized are activating transcription factor 6α -LRB- ATF6α -RRB- and ATF6β/cyclic adenosine monophosphate response element binding protein -LRB- CREB -RRB- - related protein -LRB- CREB-RP -RRB- / G13 , the protein kinase eukaryotic translation initiation factor 2α -LRB- eIF2α -RRB- kinase 3 -LRB- EIF2AK3 -RRB- / pancreatic eIF2α kinase -LRB- PEK -RRB- / PKR-like ER kinase -LRB- PERK -RRB- , and the protein kinase-endoribonucleases ER to nucleus signaling 1α -LRB- ERN1α -RRB- / inositol-requiring 1α -LRB- IRE1α -RRB- and IRE1β -LRB- Fig. 4 -RRB- . Several ATF6 paralogs , CREB3 , CREB4 , CREB-H , box B binding factor 2 -LRB- BBF2 -RRB- , and old astrocyte specifically induced substance -LRB- OASIS -RRB- have recently been described . In unstressed cells , these stress sensors are bound to BiP and kept in an inactive state . Upon ER stress , BiP is released coinciding with their activation . Whether BiP release is triggered by sequestration by unfolded proteins , conformational changes in the ER luminal domains , or reactivation of the BiP ATPase cycle is currently being debated -LRB- Kimata et al. 2004 ; Shen et al. 2005 -RRB- . ER stress also alters interactions of pro- and antiapoptotic Bcl-2 proteins at the ER membrane . These proteins regulate ER luminal Ca2 + homeostasis , ER morphology , ER stress signaling , and apoptosis induced in response to ER stress -LRB- Hetz 2007 -RRB- . Fig. 4Signal transduction pathway in the UPR . In the human UPR caspase 4 substitutes for caspase 12 . In yeast and filamentous fungi , the IRE1-XBP-1 -LRB- Hac1p/HACA -RRB- pathway is the only known UPR signal transduction pathway . -LSB- Reprinted in modified form with permission from Bentham Science Publishers from Schröder M , Kaufman , RJ -LRB- 2006 -RRB- Divergent roles of IRE1α and PERK in the unfolded protein response . Curr Mol Med 6:5 -- 36 -RSB- BiP release from ATF6 induces translocation of ATF6 to the Golgi complex where its bZIP transcription factor domain is proteolytically released from the Golgi membrane by site 1 and 2 proteases -LRB- S1P and S2P -RRB- -LRB- Fig. 4 -RRB- . This cytosolic fragment translocates to the nucleus and induces expression of chaperone genes -LRB- Strudwick and Schröder 2007 -RRB- . The functions of the ATF6 paralogs in the ER stress response is less well understood . OASIS contributes to activation of the BiP gene -LRB- Kondo et al. 2005 -RRB- , and ATF6 · CREB-H heterodimers activate inflammatory genes -LRB- Zhang et al. 2006a -RRB- . BiP release from PERK triggers oligomerization and activation of the protein kinase domain of PERK . PERK phosphorylates the bZIP transcription factor nuclear factor erythroid 2 -LRB- NF-E2 -RRB- related factor 2 -LRB- NRF2 -RRB- and eIF2α -LRB- Strudwick and Schröder 2007 -RRB- -LRB- Fig. 4 -RRB- . NRF2 phosphorylation induces its translocation to the nucleus , where a NRF2 · ATF4 heterodimer activates transcription of antioxidant response genes -LRB- Harding et al. 2003 -RRB- to counteract ROS formation by repeated folding attempts of nascent polypeptide chains in ER stressed cells . eIF2α phosphorylation attenuates general translation and decreases the influx of nascent unfolded polypeptide chains into the ER . It also clears short-lived proteins from the cell , i.e. D-type cyclins , resulting in cell cycle arrest in G1 phase , and of inhibitors of nuclear factor κB -LRB- NF-κB -RRB- -LRB- IκB -RRB- , activating the pro-inflammatory transcription factor NF-κB . eIF2α phosphorylation also induces preferential translation of capped mRNAs containing several short upstream open reading frames -LRB- uORFs -RRB- and cap-independent translation of mRNA via internal ribosomal entry sites -LRB- IRES -RRB- . Currently , the only known mRNA whose translation is stimulated by ER stress is the mRNA encoding the bZIP transcription factor ATF4 . ATF4 induces expression of CCAAT enhancer binding protein -LRB- C/EBP -RRB- homologous protein -LRB- CHOP -RRB- / CHOP-10 / growth arrest and DNA damage-inducible gene 153 -LRB- GADD153 -RRB- . CHOP represses transcription of anti-apoptotic BCL-2 -LRB- McCullough et al. 2001 -RRB- and induces expression of pro-apoptotic tribbles-related protein 3 -LRB- TRB3 -RRB- -LRB- Ohoka et al. 2005 -RRB- . Translational attenuation by PERK is transient and countered by several eIF2α phosphatases . The regulatory subunit of protein phosphatase 1 -LRB- PP1 -RRB- , GADD34 , is induced late in ER stress by ATF4 , and targets PP1 to eIF2α . Upon BiP release , IRE1 autophosphorylates in trans and activates its endoribonuclease domain -LRB- Strudwick and Schröder 2007 -RRB- . Activated IRE1 cleaves exon-intron junctions in the mRNAs encoding the bZIP transcription factors homologous to ATF/CREB1 protein -LRB- Hac1p -RRB- in yeast , HACA in filamentous fungi , and X box-binding protein 1 -LRB- XBP-1 -RRB- / hepatocarcinogenesis-related transcription factor -LRB- HTF -RRB- / tax-responsive element binding protein 5 -LRB- TREB5 -RRB- in metazoans -LRB- Fig. 4 -RRB- . In yeast , the HAC1 exons are ligated by transfer RNA ligase . Both transcription factors activate expression of genes encoding ER-resident molecular chaperones , protein foldases , and genes encoding proteins involved in ERAD . Hac1p and XBP-1 also stimulate phospholipid biosynthesis . Spliced Hac1p represses genes under control of the transcriptional regulator unscheduled meiosis 6 protein -LRB- Ume6p -RRB- , including the early meiotic genes and a large array of genes involved in carbon and nitrogen metabolism . This function of spliced Hac1p may explain the slow growth phenotype of yeast cells overexpressing Ire1p or spliced Hac1p -LRB- Mori et al. 2000 -RRB- . Attenuation of metabolism by Hac1p in yeast may substitute for attenuation of general translation by PERK . XBP-1 activation is delayed compared to ATF6 and PERK activation -LRB- Yoshida et al. 2003 -RRB- , allowing for an early folding only phase in the UPR followed by a folding and degradation phase . In mammals , phosphorylated IRE1 sequesters tumor necrosis factor receptor associated factor 2 -LRB- TRAF2 -RRB- from pro-caspase 12 , resulting in clustering and activation of this caspase and initiation of apoptosis -LRB- Strudwick and Schröder 2007 -RRB- -LRB- Fig. 4 -RRB- . TRAF2 recruits apoptosis signal-regulating kinase 1 -LRB- ASK1 -RRB- and IκB kinase to IRE1 . ASK1 activates the mitogen-activated protein -LRB- MAP -RRB- kinases p38 and ju-nana -LRB- jun -RRB- N-terminal kinases -LRB- JNKs -RRB- . p38 and JNKs phosphorylate and potentiate pro-apoptotic transcription factors , i.e. CHOP and c-Jun , and phosphorylate and inhibit anti-apoptotic Bcl-2 and Bcl-xL to induce apoptosis -LRB- Szegezdi et al. 2006 -RRB- . Inhibition of JNK phosphatases by ROS formed in oxidative protein folding also contributes to JNK activation -LRB- Kamata et al. 2005 -RRB- . Association of the pro-apoptotic Bcl-2 family proteins Bax and Bak with the cytosolic portion of IRE1 is required for efferent IRE1 signaling , i.e. XBP-1 mRNA splicing and JNK1 activation -LRB- Hetz et al. 2006 -RRB- . Engineering of the UPR Engineering of the UPR holds promise to increase concentrations of several chaperones in a functionally meaningful ratio , thus preventing inadvertent conversion of chaperone foldases into holdases , or even inactivating chaperones because of lack of concomitant increases in co-chaperones and co-factors . The UPR is as much a survival response as it is a response to boost protein secretion . Clearly , some aspects of the physiological UPR are not desirable in an expression system , for example inhibition of general translation by PERK , stimulation of ERAD and apoptosis . Overexpression of spliced Hac1p or its filamentous fungi homolog HACA increased heterologous protein secretion in Saccharomyces cerevisiae and Aspergillus niger -LRB- Valkonen et al. 2003a , b -RRB- . In Chinese hamster ovary -LRB- CHO -RRB- - K1 cells XBP-1 overexpression improved secretion of several heterologous proteins -LRB- Ku et al. 2007 ; Tigges and Fussenegger 2006 -RRB- . In yeast and filamentous fungi , secretion of several heterologous proteins was not improved or even decreased by overexpression of Hac1p -LRB- Gasser et al. 2006 ; Rakestraw and Wittrup 2005 ; Valkonen et al. 2003a -RRB- . One drawback of some of these studies is that wild-type -LRB- WT -RRB- cells with a functional UPR are compared to WT cells overexpressing spliced Hac1p , which may explain why in some cases only marginal differences between the two cell types have been observed . The more consistent effect of spliced XBP-1 overexpression on protein secretion -LRB- Ku et al. 2007 ; Tigges and Fussenegger 2006 -RRB- may be caused by a preferential activation of UPR signaling pathways that stimulate cellular functions beneficial for heterologous protein secretion , i.e. increased chaperone gene expression . This increased ER-resident chaperone machinery attenuates all UPR signaling , and most importantly , those UPR signaling pathways likely to decrease protein production , for example general translation inhibition by PERK , stimulation of ERAD and of apoptosis -LRB- Özcan et al. 2004 -RRB- . Conclusions Engineering of chaperone systems by overexpressing a single component of the ER-resident protein folding machinery has overall yielded mixed results . Our basic understanding of protein folding in the ER is still incomplete . Addressing these open questions should underpin engineering approaches to improve the performance of chaperone systems . A more detailed understanding of chaperone function and regulation should inform future work to improve chaperone systems . Co-expression of different holdases or targeting of heterologous or cytosolic holdases to the ER may yield more consistent improvements for different heterologous proteins . The function of overexpressed chaperones may not be the same as at their normal physiological concentrations , because of the lack of a corresponding increase in co-chaperones and co-factors . To improve foldase function , concomitant elevation of BiP , its co-chaperones , and ATP levels should be attempted . Engineering of the UPR suggests that mimicking an UPR by expression of its activated signaling molecules does not consistently improve productivities . Dissection of UPR signaling activities may be necessary to improve heterologous protein secretion . To place engineering of the UPR on an informed basis , we need to understand the UPR in more detail . It is still not clear what the most upstream events in activation of the UPR are , how UPR signaling integrates into cellular signaling , and how the UPR decides between a prosurvival and an apoptotic response to ER stress . Cell engineering also needs to address the potential problem that increased oxidative protein folding may be inherently toxic to cells , because of elevated cellular ROS levels .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O

molecular chaperone
protein folding
unfolded protein response

endoplasmic reticulum associated protein degradation


Differential

activities

of

the

core

planar

polarity

proteins

during

Drosophila

wing

patterning

During

planar

polarity

patterning

of

the

Drosophila

wing

a

core

group

of

planar

polarity

genes

has

been

identified

which

acts

downstream

of

global

polarity

cues

to

locally

coordinate

cell

polarity

and

specify

trichome

production

at

distal

cell

edges

These

genes

encode

protein

products

that

assemble

into

asymmetric

apicolateral

complexes

that

straddle

the

proximodistal

junctional

region

between

adjacent

cells

We

have

carried

out

detailed

genetic

analysis

experiments

analysing

the

requirements

of

each

complex

component

for

planar

polarity

patterning

We

find

that

the

three

transmembrane

proteins

at

the

core

of

the

complex

Frizzled

Strabismus

and

Flamingo

are

required

earliest

in

development

and

are

the

only

components

needed

for

intercellular

polarity

signalling

Notably

cells

that

lack

both

Frizzled

and

Strabismus

are

unable

to

signal

revealing

an

absolute

requirement

for

both

proteins

in

cell

cell

communication

In

contrast

the

cytoplasmic

components

Dishevelled

Prickle

and

Diego

are

not

needed

for

intercellular

communication

These

factors

contribute

to

the

cell

cell

propagation

of

polarity

most

likely

by

promotion

of

intracellular

asymmetry

Interestingly

both

local

polarity

propagation

and

trichome

placement

occur

normally

in

mutant

backgrounds

where

asymmetry

of

polarity

protein

distribution

is

undetectable

suggesting

such

asymmetry

is

not

an

absolute

requirement

for

any

of

the

functions

of

the

core

complex

Introduction

The

term

planar

polarity

was

first

used

to

describe

the

polarisation

of

structures

within

the

plane

of

the

insect

cuticle

-LRB-

Nübler-Jung

et

al.

-RRB-

however

the

phenomenon

is

widespread

in

nature

-LRB-

reviewed

in

Klein

and

Mlodzik

-RRB-

Genetic

analysis

particularly

in

Drosophila

has

identified

a

planar

polarity

or

PCP

-LRB-

planar

cell

polarity

-RRB-

pathway

dependent

on

the

function

of

Frizzled

-LRB-

Fz

-RRB-

family

receptors

Interestingly

not

only

are

elements

of

this

pathway

conserved

throughout

the

animal

kingdom

but

it

is

also

required

for

developmental

patterning

processes

that

are

distinct

from

planar

polarity

such

as

polarised

cell

rearrangements

during

vertebrate

gastrulation

-LRB-

Wallingford

et

al.

-RRB-

To

date

planar

polarity

patterning

has

been

best

studied

in

the

Drosophila

wing

which

provides

a

simple

model

in

which

each

cell

becomes

coordinately

polarised

and

produces

a

single

distally

pointing

trichome

-LRB-

Fig.

-RRB-

It

is

widely

considered

that

this

pattern

is

produced

by

three

tiers

of

gene

activity

-LRB-

Tree

et

al.

Klein

and

Mlodzik

Strutt

and

Strutt

-RRB-

At

the

top

of

the

hierarchy

the

type

II

transmembrane

protein

Four-jointed

-LRB-

Fj

-RRB-

and

the

atypical

cadherins

Dachsous

-LRB-

Ds

-RRB-

and

Fat

-LRB-

Ft

-RRB-

act

-LRB-

probably

with

other

unidentified

factors

-RRB-

to

provide

a

long-range

-LRB-

or

global

-RRB-

patterning

cue

across

the

axis

of

the

tissue

-LRB-

Adler

et

al.

Zeidler

et

al.

Strutt

and

Strutt

Ma

et

al.

-RRB-

In

a

manner

which

is

not

understood

but

is

possibly

dependent

on

widerborst

gene

function

-LRB-

Hannus

et

al.

-RRB-

this

long-range

cue

is

thought

to

be

interpreted

by

the

middle

tier

of

genes

which

include

fz

and

a

number

of

other

factors

known

as

the

core

polarity

genes

-LRB-

Shulman

et

al.

-RRB-

The

final

tier

consists

of

tissue-specific

effectors

which

modulate

cellular

behaviours

such

as

polarisation

of

the

cytoskeleton

and

transcription

in

response

to

activity

of

components

of

the

core

The

definition

of

the

core

polarity

proteins

is

somewhat

fluid

but

was

originally

used

to

refer

to

factors

that

act

together

with

Fz

in

all

tissues

examined

in

Drosophila

A

notable

property

of

Fz

during

planar

polarity

patterning

is

that

it

adopts

an

asymmetric

subcellular

localisation

in

polarising

cells

for

instance

in

the

wing

becoming

localised

to

the

junctional

zone

at

the

distal

cell

edge

-LRB-

Strutt

-RRB-

Five

other

proteins

that

act

with

Fz

also

adopt

asymmetric

localisations

either

at

the

proximal

or

distal

edges

of

wing

cells

and

loss

of

any

one

of

these

proteins

prevents

the

distal

localisation

of

Fz

As

these

proteins

colocalise

to

junctions

with

Fz

and

are

required

for

Fz

localisation

it

seems

reasonable

to

regard

them

as

the

core

They

consist

of

the

multidomain

cytoplasmic

protein

Dishevelled

-LRB-

Dsh

-RRB-

and

the

ankyrin

repeat

protein

Diego

-LRB-

Dgo

-RRB-

that

localise

distally

with

Fz

-LRB-

Axelrod

Das

et

al.

-RRB-

the

fourpass

transmembrane

protein

Strabismus

-LRB-

Stbm

also

known

as

Van

Gogh

-LSB-

Vang

-RSB-

-RRB-

and

the

LIM-domain

protein

Prickle

that

localise

proximally

-LRB-

Bastock

et

al.

Tree

et

al.

-RRB-

and

the

sevenpass

transmembrane

cadherin

Flamingo

-LRB-

Fmi

also

known

as

Starry

Night

-LSB-

Stan

-RSB-

-RRB-

that

localises

both

proximally

and

distally

-LRB-

Chae

et

al.

Usui

et

al.

-RRB-

-LRB-

Fig.

-RRB-

We

note

that

by

this

definition

the

Gαo

subunit

encoded

by

the

brokenheart

gene

may

also

be

regarded

as

a

component

of

the

core

-LRB-

Katanaev

et

al.

-RRB-

but

this

requires

further

investigation

Fz

is

thought

to

perform

at

least

three

functions

in

planar

polarity

patterning

The

first

is

to

receive

long-range

pattering

information

from

upstream

cues

for

instance

provided

by

the

activities

of

Fj/Ds/Ft

Experiments

analysing

the

temporal

requirements

of

fz

and

ds

suggest

that

such

coupling

may

occur

around

to

h

of

pupal

life

-LRB-

Strutt

and

Strutt

Matakatsu

and

Blair

-RRB-

Recent

models

have

suggested

that

this

information

could

be

provided

either

by

generation

of

a

gradient

of

Fz

activity

across

the

whole

axis

of

the

wing

or

alternatively

via

generation

of

a

gradient

of

Fz

activity

across

the

axis

of

individual

cells

-LRB-

Lawrence

et

al.

Amonlirdviman

et

al.

-RRB-

Notably

there

is

currently

no

evidence

that

other

components

of

the

core

are

involved

in

this

coupling

Second

Fz

is

involved

in

a

process

of

cell

cell

communication

that

locally

coordinates

cell

polarity

-LRB-

Adler

et

al.

Ma

et

al.

Lawrence

et

al.

-RRB-

and

also

occurs

after

h

of

pupal

life

-LRB-

Strutt

and

Strutt

-RRB-

Historically

models

to

explain

this

coordination

have

invoked

the

production

of

a

diffusible

ligand

for

Fz

-LRB-

Park

et

al.

Zheng

et

al.

Adler

et

al.

-RRB-

However

more

recent

models

based

on

the

observation

of

core

polarity

protein

localisation

to

cell

junctions

have

suggested

that

cell

cell

signalling

is

contact-dependent

-LRB-

Tree

et

al.

Lawrence

et

al.

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

Generally

it

has

been

assumed

that

all

components

of

the

core

act

with

Fz

in

local

coordination

of

polarity

but

the

exact

roles

of

each

protein

have

not

been

defined

The

third

function

of

Fz

is

to

provide

a

subcellular

cue

for

trichome

growth

apparently

via

its

localisation

to

the

distal

cell

edge

-LRB-

Wong

and

Adler

Strutt

-RRB-

In

the

absence

of

Fz

or

several

other

core

components

trichomes

form

in

the

cell

centre

Provision

of

Fz

activity

after

h

of

pupal

life

is

sufficient

to

permit

asymmetric

localisation

and

polarised

trichome

growth

-LRB-

Strutt

and

Strutt

-RRB-

however

distal

polarity

is

lost

presumably

due

to

disruption

of

earlier

fz

functions

As

all

core

components

asymmetrically

localise

together

with

Fz

prior

to

trichome

formation

it

is

tempting

to

conclude

that

all

are

required

for

trichome

placement

but

this

has

not

been

definitively

demonstrated

Asymmetric

localisation

of

the

core

components

only

becomes

clearly

visible

during

pupal

life

by

about

h

of

pupal

life

-LRB-

but

has

also

been

observed

earlier

in

development

see

Classen

et

al.

-RRB-

and

hence

it

has

been

suggested

that

this

probably

follows

the

cell

cell

communication

phase

-LRB-

Strutt

and

Strutt

Lawrence

et

al.

-RRB-

However

other

workers

have

argued

that

asymmetric

complex

formation

may

occur

progressively

over

a

longer

period

of

pupal

life

and

be

intrinsically

required

for

cell

cell

communication

and

local

coordination

of

polarity

-LRB-

Tree

et

al.

Amonlirdviman

et

al.

-RRB-

In

this

context

it

is

important

to

consider

that

the

spatial

relationships

observed

during

asymmetric

complex

formation

-LRB-

Fz

Dsh

Dgo

and

Fmi/Stan

colocalising

at

distal

cell

edges

Stbm/Vang

Pk

and

Fmi/Stan

at

proximal

edges

-RRB-

may

not

necessarily

reflect

earlier

functional

relationships

Notably

associations

have

also

been

reported

between

Dsh

and

Pk

-LRB-

Tree

et

al.

Jenny

et

al.

-RRB-

Dsh

and

Stbm/Vang

-LRB-

Bastock

et

al.

Jenny

et

al.

-RRB-

Dgo

and

Pk

-LRB-

Das

et

al.

-RRB-

and

Dgo

and

Stbm/Vang

-LRB-

Das

et

al.

-RRB-

In

this

manuscript

we

address

three

key

issues

First

which

components

of

the

core

act

together

with

Fz

during

the

different

planar

polarity

patterning

processes

Second

are

the

spatial

relationships

seen

during

the

later

phase

of

asymmetric

localisation

also

relevant

during

the

phase

of

cell

cell

communication

and

local

coordination

of

polarity

Third

is

asymmetric

core

protein

localisation

absolutely

required

for

planar

polarity

patterning

Materials

and

methods

Fly

strains

and

genetics

Alleles

and

transgenes

used

are

described

in

FlyBase

except

where

noted

Temporal

rescue

of

polarity

phenotypes

in

the

wing

and

eye

was

carried

out

and

analysed

as

described

-LRB-

Strutt

and

Strutt

-RRB-

Actin

fz-EYFP

and

Actin

stbm-EYFP

have

been

described

-LRB-

Strutt

Strutt

et

al.

-RRB-

Actin

dsh-ECFP

Actin

fmi-FLAG

Actin

pkpk

sev-stbm

and

sev-pksple

were

constructed

as

previously

-LRB-

Strutt

and

Strutt

-RRB-

Note

that

the

pk

locus

produces

two

protein

isoforms

of

which

the

Pk

variant

is

sufficient

for

wing

patterning

and

the

Sple

variant

is

sufficient

for

eye

patterning

-LRB-

Gubb

et

al.

-RRB-

For

double

mutant

clones

rescue

of

fz

activity

on

the

X

and

was

provided

by

Arm-fz-EGFP

transgenes

-LRB-

Strutt

-RRB-

and

rescue

of

stbm/Vang

activity

on

the

X

was

provided

by

an

Actin-stbm-EYFP

transgene

fz

stbm

twinclones

were

generated

by

inducing

clones

of

Arm-fz-EGFP

in

a

fz

background

resulting

in

cells

homozygous

for

Arm-fz-EGFP

juxtaposed

to

twinspot

cells

lacking

the

transgene

Clones

in

the

wing

were

generally

induced

using

Ubx-FLP

kindly

provided

by

Jürgen

Knoblich

Exact

genotypes

used

are

as

follows

Figure

Temporal

rescue

of

stbm/Vang

in

wing

w

Act-FRT-polyA-FRT-stbm-EYFP

Temporal

rescue

of

pkpk-sple

in

wing

w

Act-FRT-polyA-FRT-pk

stbm/Vang

phenotype

in

eye

w

P

-LSB-

w

-RSB-

Rescue

of

stbm/Vang

phenotype

in

eye

by

sev-stbm

w

P

-LSB-

w

-RSB-

phenotype

in

eye

cn

cn

Rescue

of

pkpk-sple

phenotype

in

eye

by

sev-pksple

w

cn

Genotypes

shown

in

graph

in

-LRB-

S

-RRB-

w

P

-LSB-

w

-RSB-

w

P

-LSB-

w

-RSB-

w

P

-LSB-

w

-RSB-

Genotypes

shown

in

graph

in

-LRB-

T

-RRB-

cn

w

cn

sevE-sevP-pksple

-LSB-

-RSB-

w

cn

sevE-sevP-pksple

-LSB-

-RSB-

Figure

fz

clones

using

rescuing

transgene

on

w

Arm-fz-EGFP

Arm-lacZ

Ubx-FLP

stbm/Vang

clones

y

w

Ubx-FLP

Arm-lacZ

stbm/Vang

fz

double

clones

w

Arm-fz-EGFP

Ubx-FLP

stbm/Vang

and

fz

twin

clones

w

Arm-lacZ

Ubx-FLP

fmi/stan

clones

y

w

Ubx-FLP

Arm-lacZ

fmi/stan

fz

double

clones

w

Arm-fz-EGFP

Arm-lacZ

Ubx-FLP

stbm/Vang

fmi/stan

double

clones

y

w

Ubx-FLP

Arm-lacZ

Figure

clones

y

w

Arm-lacZ

dsh

fz

double

clones

y

w

Arm-fz-EGFP

Ubx-FLP

dsh

stbm/Vang

double

clones

y

w

Act-FRT-polyA-FRT-stbm-EYFP

Ubx-FLP

pkpk-sple

clones

y

w

Ubx-FLP

Arm-lacZ

pkpk-sple

fz

double

clones

w

Arm-fz-EGFP

Ubx-FLP

pkpk-splestbm/Vang

double

clones

y

w

Ubx-FLP

Arm-lacZ

Figure

stbm/Vang

dgo

double

clones

y

w

Ubx-FLP

Arm-lacZ

pkpk-spledgo

double

clones

y

w

Ubx-FLP

Arm-lacZ

pkpk-spledgo

fz

triple

clones

w

Arm-fz-EGFP

Ubx-FLP

Figure

stbm/Vang

overexpression

in

fz

background

y

w

Act-FRT-y

UAS-lacZ

UAS-stbm/Df

-LRB-

-RRB-

fz-EGFP

overexpression

in

stbm/Vang

background

w

Act-FRT-y

UAS-fz-EGFP

fz-EGFP

overexpression

in

pkpk-sple

background

w

Act-FRT-y

UAS-fz-EGFP

fz-EGFP

overexpression

in

pkpk-sple

background

w

UAS-fz-EGFP

fz-EGFP

overexpression

in

w

UAS-lacZ

UAS-fz

fz-EGFP

overexpression

under

control

in

wings

containing

clones

y

w

w

w

UAS-fz-EGFP

Ubx-FLP

Figure

Act-fz-EYFP

expression

in

pkpk-sple

background

w

Act-FRT-polyA-FRT-fz-EYFP

Act-fz-EYFP

expression

in

background

w

Act-FRT-polyA-FRT-fz-EYFP

dgo

clones

y

w

Ubx-FLP

Arm-lacZ

pkpk-sple

clones

y

w

Ubx-FLP

Arm-lacZ

pkpk-spledgo

double

clones

y

w

Ubx-FLP

Arm-lacZ

clones

y

w

Arm-lacZ

Supplementary

Figure

Temporal

rescue

of

fz

y

w

Act-FRT-polyA-FRT-fz-EYFP

Temporal

rescue

of

fmi/stan

y

w

Act-FRT-polyA-FRT-fmi-FLAG

UAS-fmi

and

UAS-fmi

provide

rescue

of

fmi

activity

in

the

embryonic

nervous

system

-LRB-

Usui

et

al.

-RRB-

Temporal

rescue

of

dsh

w

Act

FRT-poly-FRT-dsh-ECFP

Supplementary

Figure

w

stbm

stbm

Act-FRT-polyA-FRT-stbm-EYFP

y

w

Act-FRT-polyA-FRT-fmi-FLAG

UAS-fmi

w

dsh

Y

Act

FRT-poly-FRT-dsh-ECFP

w

pk

pk

Act-FRT-polyA-FRT-pk

Supplementary

Figure

stbm/Vang

clones

w

P

-LSB-

w

-RSB-

stbm/Vang

clones

rescue

by

sev-stbm

w

P

-LSB-

w

-RSB-

Note

that

and

have

been

molecularly

characterised

and

are

thought

to

be

alleles

on

the

basis

of

being

unable

to

give

rise

to

functional

proteins

-LRB-

Jones

et

al.

Wolff

and

Rubin

Wehrli

and

Tomlinson

Usui

et

al.

Gubb

et

al.

Feiguin

et

al.

-RRB-

contains

a

missense

mutation

that

generates

an

amorphic

mutation

in

the

wing

by

genetic

criteria

-LRB-

Usui

et

al.

-RRB-

contains

a

nonsense

mutation

that

gives

rise

to

a

truncated

protein

that

has

been

characterised

as

amorphic

in

the

wing

-LRB-

Jones

et

al.

-RRB-

has

not

been

molecularly

characterised

but

has

been

defined

by

genetic

criteria

to

be

amorphic

in

the

wing

-LRB-

Taylor

et

al.

-RRB-

contains

a

missense

mutation

in

the

DEP

domain

which

has

been

reported

to

be

a

strong

mutation

for

planar

polarity

functions

of

the

gene

-LRB-

Perrimon

and

Mahowald

Axelrod

et

al.

Boutros

et

al.

-RRB-

Histology

Pupal

wings

were

processed

for

immunofluorescence

and

imaged

as

previously

-LRB-

Strutt

-RRB-

Primary

antibodies

used

for

experiments

or

confirmation

of

genotypes

were

mouse

monoclonal

anti-βgal

-LRB-

Promega

-RRB-

rabbit

anti-βgal

-LRB-

Cappel

-RRB-

rabbit

anti-GFP

-LRB-

Abcam

-RRB-

mouse

monoclonal

anti-Fmi

-LRB-

DSHB

Usui

et

al.

-RRB-

rabbit

anti-Pk

-LRB-

Tree

et

al.

-RRB-

rabbit

anti-Stbm

-LRB-

Rawls

and

Wolff

-RRB-

rat

anti-Dsh

-LRB-

Shimada

et

al.

-RRB-

and

rabbit

anti-Dgo

-LRB-

Feiguin

et

al.

-RRB-

Actin

was

visualised

using

Texas-Red-conjugated

phalloidin

-LRB-

Molecular

Probes

-RRB-

Adult

wings

were

mounted

in

GMM

and

eye

sections

were

prepared

as

described

-LRB-

Tomlinson

and

Ready

-RRB-

Results

Differing

temporal

requirements

of

the

core

polarity

proteins

during

wing

development

We

previously

analysed

the

temporal

requirements

of

fz

for

planar

polarity

patterning

in

the

wing

by

rescuing

the

phenotype

of

fz

mutant

flies

using

an

inducible

fz-expressing

transgene

-LRB-

Strutt

and

Strutt

-RRB-

Expression

of

the

transgene

is

activated

at

different

times

during

pupal

development

by

administration

of

a

heat-shock

allowing

determination

of

the

latest

timepoint

that

gene

expression

is

sufficient

to

permit

normal

patterning

These

studies

found

no

requirement

for

fz

function

prior

to

h

after

prepupa

formation

-LRB-

APF

-RRB-

Progressively

later

heat-shocks

up

to

h

APF

produced

stronger

phenotypes

that

were

qualitatively

and

quantitatively

different

from

the

reported

fz

loss-of-function

phenotype

We

classified

this

stronger

phenotype

as

ds-like

as

Fz

protein

was

still

localising

at

cell

edges

and

specifying

the

site

of

trichome

formation

but

due

to

a

loss

of

non-autonomous

coordination

of

polarity

Fz

localisation

was

seen

in

a

swirling

pattern

rather

than

uniformly

at

distal

cell

edges

-LRB-

Strutt

and

Strutt

-RRB-

Heat-shocks

after

h

APF

resulted

in

the

reported

fz

loss-of-function

phenotype

consistent

with

this

being

produced

by

loss

of

the

later

autonomous

function

that

places

trichomes

at

the

cell

edge

As

the

core

polarity

gene

stbm/Vang

shows

similar

phenotypes

to

fz

exhibiting

both

strong

domineering

non-autonomous

effects

on

trichome

polarity

and

being

required

for

trichome

placement

at

the

cell

edge

-LRB-

Taylor

et

al.

-RRB-

we

considered

it

a

good

candidate

for

sharing

common

functions

with

fz

Using

the

same

methodology

we

analysed

its

timecourse

of

requirement

in

wing

patterning

-LRB-

Figs.

I

-RRB-

In

common

with

fz

stbm/Vang

is

not

required

prior

to

h

APF

but

then

shows

progressively

stronger

phenotypes

when

induced

between

and

h

APF

with

induction

at

h

APF

mimicking

the

normal

loss-of-function

phenotype

-LRB-

seen

when

no

heat-shock

is

administered

Fig.

-RRB-

For

comparison

we

repeated

our

analysis

of

the

timecourse

of

fz-requirement

-LRB-

Supplementary

Fig.

-RRB-

but

this

time

using

the

molecularly

characterised

allele

-LRB-

Jones

et

al.

-RRB-

This

gave

the

same

timecourse

as

observed

for

stbm/Vang

although

generally

with

slightly

stronger

phenotypes

being

observed

Next

we

analysed

the

temporal

requirement

of

the

core

polarity

gene

pk

which

produces

a

protein

that

colocalises

with

Stbm/Vang

at

the

proximal

cell

edge

and

which

has

been

implicated

in

cell

cell

coordination

of

planar

polarity

-LRB-

Tree

et

al.

-RRB-

Interestingly

induction

of

pk

expression

as

late

as

h

APF

resulted

in

only

negligible

polarity

defects

in

the

adult

wing

with

induction

at

h

and

h

still

providing

partial

rescue

of

pk

function

-LRB-

Figs.

N

-RRB-

These

results

indicate

that

whereas

stbm/Vang

shares

an

early

requirement

with

fz

in

the

wing

pk

has

only

a

relatively

late

function

We

further

extended

these

results

by

investigating

the

requirements

of

the

other

two

core

components

fmi/stan

and

dsh

-LRB-

Supplementary

Figs.

C

-RRB-

To

circumvent

the

embryonic

lethality

of

dsh

alleles

we

analysed

rescue

of

the

strong

planar

polarity

phenotype

of

the

viable

allele

-LRB-

Perrimon

and

Mahowald

-RRB-

-LRB-

the

core

component

dgo

was

not

examined

as

the

adult

wing

phenotype

is

too

subtle

for

this

approach

to

be

feasible

Feiguin

et

al.

-RRB-

Induction

of

fmi/stan

expression

between

and

h

APF

resulted

in

progressively

stronger

phenotypes

that

differ

from

the

loss-of-function

phenotype

-LRB-

Supplementary

Fig.

-RRB-

as

observed

for

fz

and

stbm/Vang

Conversely

induction

of

dsh

at

to

h

APF

resulted

in

relatively

minor

defects

although

later

induction

revealed

a

strong

requirement

for

dsh

function

after

h

APF

-LRB-

Supplementary

Fig.

-RRB-

Hence

fmi/stan

appears

to

share

early

requirements

with

fz

and

stbm/Vang

whereas

dsh

exhibits

later

temporal

requirements

However

we

can

not

rule

out

the

possibility

that

the

allele

exhibits

residual

activity

in

planar

polarity

which

might

contribute

to

the

apparently

later

requirement

For

all

genotypes

early

transgene

induction

can

rescue

indicating

that

the

transgenes

provide

appropriate

levels

of

expression

throughout

the

wing

Consistent

with

this

almost

all

cells

express

detectable

protein

after

transgene

induction

-LRB-

Supplementary

Fig.

-RRB-

Furthermore

without

induction

we

observe

the

expected

loss-of-function

phenotype

seen

in

the

absence

of

the

transgene

indicating

that

our

results

are

unlikely

to

be

due

to

leaky

expression

from

the

transgenes

We

tested

whether

the

differences

might

be

due

to

transmembrane

proteins

taking

longer

to

be

synthesised

and

targeted

to

the

appropriate

subcellular

sites

however

we

found

that

after

induction

both

Fz-EYFP

and

Dsh-ECFP

show

the

appearance

of

junctional

staining

within

h

-LRB-

data

not

shown

-RRB-

Differing

temporal

requirements

of

the

core

polarity

proteins

during

eye

development

We

also

find

a

common

early

requirement

for

fz

and

stbm/Vang

in

the

eye

We

previously

distinguished

between

early

and

late

activities

of

fz

in

the

eye

by

expressing

fz

under

control

of

the

sevenless

promoter

which

is

not

active

until

the

time

of

photoreceptor

differentiation

-LRB-

Strutt

and

Strutt

-RRB-

Providing

fz

activity

only

at

the

time

of

photoreceptor

differentiation

resulted

in

defects

in

the

dorsoventral

polarity

of

ommatidia

indicating

that

fz

activity

is

specifically

required

prior

to

photoreceptor

differentation

for

correct

specification

of

dorsoventral

polarity

However

lack

of

fz

activity

after

photoreceptor

differentiation

results

in

randomisation

of

all

aspects

of

ommatidial

polarity

including

both

dorsoventral

and

anteroposterior

polarity

and

rotation

Hence

fz

shows

two

phases

of

requirement

during

eye

development

an

early

phase

needed

just

for

dorsoventral

patterning

and

a

later

phase

required

for

dorsoventral

and

anteroposterior

polarity

and

rotation

of

ommatidia

In

contrast

we

showed

that

ommatidial

polarity

and

rotation

defects

within

mutant

tissue

can

be

rescued

completely

by

expression

of

sev-dsh

indicating

that

dsh

does

not

share

the

early

dorsoventral

patterning

function

with

fz

but

nevertheless

is

required

for

the

later

phase

of

activity

We

have

now

extended

this

work

to

stbm/Vang

and

pk

Rescuing

the

phenotype

of

stbm/Vang

in

the

eye

by

expression

of

sev-stbm

reduces

general

polarity

and

rotation

defects

but

reveals

an

underlying

randomisation

of

dorsoventral

polarity

-LRB-

Figs.

P

S

-RRB-

indicating

that

stbm/Vang

shares

with

fz

an

early

dorsoventral

patterning

function

Conversely

the

pk

phenotype

is

almost

completely

rescued

by

a

sev-pksple

transgene

-LRB-

Figs.

R

T

-RRB-

which

expresses

the

Sple

isoform

of

the

Pk

protein

which

is

specifically

required

for

eye

patterning

-LRB-

Gubb

et

al.

-RRB-

Thus

pk

like

dsh

does

not

exhibit

an

early

patterning

function

in

eye

development

Interestingly

while

investigating

the

functions

of

stbm/Vang

in

the

eye

we

found

that

stbm/Vang

clones

also

show

equatorial

non-autonomy

of

the

polarity

phenotype

In

a

number

of

cases

we

observed

dorsoventral

polarity

inversions

in

ommatidia

on

the

equatorial

sides

of

clones

in

which

all

photoreceptors

of

the

ommatidium

retain

stbm/Vang

activity

-LRB-

Supplementary

Fig.

-RRB-

This

is

consistent

with

the

observed

non-autonomy

of

clones

in

the

wing

-LRB-

Taylor

et

al.

-RRB-

However

a

previous

analysis

of

over

misoriented

ommatidia

on

the

edges

of

clones

found

no

significant

evidence

of

non-autonomy

of

the

polarity

phenotype

of

stbm/Vang

-LRB-

Wolff

and

Rubin

-RRB-

Re-examination

of

this

original

data

set

in

the

light

of

our

results

again

failed

to

find

evidence

of

non-autonomy

-LRB-

T.

Wolff

personal

communication

-RRB-

The

reasons

for

this

discrepancy

are

currently

unclear

-LRB-

but

see

legend

to

Supplementary

Fig.

for

further

discussion

of

this

issue

-RRB-

Mutual

dependence

of

fz

stbm/Vang

and

fmi/stan

for

intercellular

communication

The

common

early

function

of

fz

stbm/Vang

and

fmi/stan

is

likely

to

be

either

receiving

long-range

patterning

cues

and/or

local

coordination

of

polarity

Little

is

understood

about

how

the

long-range

signal

might

be

received

rendering

this

activity

difficult

to

study

However

the

effects

of

fz

and

stbm/Vang

on

local

coordination

of

polarity

can

be

assayed

as

groups

of

cells

lacking

the

activity

of

either

gene

cause

neighbouring

cells

to

mispolarise

fz

clones

cause

neighbouring

cells

to

point

their

trichomes

towards

the

clone

-LRB-

arrow

Fig.

Gubb

and

García-Bellido

Vinson

and

Adler

-RRB-

whereas

trichomes

point

away

from

stbm/Vang

clones

-LRB-

arrow

Fig.

Taylor

et

al.

-RRB-

As

during

later

pupal

life

Fz

and

Stbm/Vang

are

seen

localised

to

adjacent

cell

boundaries

-LRB-

Strutt

Bastock

et

al.

-RRB-

it

has

been

proposed

that

polarity

coordination

requires

signals

to

pass

between

Fz

and

Stbm/Vang

expressing

cells

Some

evidence

for

this

has

been

presented

in

the

abdomen

-LRB-

Lawrence

et

al.

-RRB-

and

recent

models

for

planar

polarity

coordination

in

the

wing

are

based

on

this

hypothesis

-LRB-

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

However

there

is

no

rigorous

experimental

evidence

for

signals

passing

between

Fz

and

Stbm/Vang

expressing

cells

in

the

wing

Furthermore

if

such

signalling

does

occur

it

is

not

known

whether

signals

might

pass

monodirectionally

from

Fz

to

Stbm/Vang

monodirectionally

from

Stbm/Vang

to

Fz

or

bidirectionally

To

address

this

issue

we

generated

clones

of

cells

simultaneously

mutant

for

both

fz

and

stbm/Vang

We

reasoned

that

if

signals

pass

strictly

monodirectionally

from

Stbm/Vang

to

Fz

then

wild-type

cells

outside

of

a

clone

would

receive

the

same

aberrant

polarity

cue

from

a

stbm/Vang

fz

double

mutant

clone

as

from

a

stbm/Vang

single

mutant

clone

Thus

stbm/Vang

fz

double

mutant

clones

should

show

the

same

polarity

phenotype

as

stbm/Vang

single

mutant

clones

Conversely

if

signals

pass

strictly

from

Fz

to

Stbm/Vang

cells

outside

should

polarise

as

if

neighbouring

a

fz

clone

and

not

a

stbm/Vang

clone

In

this

case

cells

require

Fz

to

send

polarity

cues

and

cells

mutant

for

both

fz

and

stbm/Vang

provide

the

same

aberrant

polarity

cue

as

cells

mutant

for

only

fz

However

if

there

is

a

bidirectional

interaction

such

that

cells

expressing

Fz

need

to

contact

cells

expressing

Stbm/Vang

and

vice

versa

then

the

result

is

harder

to

predict

In

this

case

signal

receiving

cells

would

require

both

Fz

and

Stbm/Vang

and

similarly

signal

sending

cells

would

require

both

Fz

and

Stbm/Vang

Hence

one

possibility

is

that

clones

of

cells

doubly

mutant

for

fz

and

stbm/Vang

would

send

or

receive

no

polarity

signals

and

thus

might

have

no

effect

on

the

polarity

of

their

neighbours

A

precedent

for

this

prediction

has

been

provided

by

work

in

the

abdomen

where

experimental

results

suggest

that

cells

that

lack

Fmi/Stan

are

unable

to

send

or

receive

polarity

cues

and

in

this

case

the

neighbours

to

exhibit

normal

polarity

-LRB-

Lawrence

et

al.

-RRB-

However

it

is

also

possible

that

a

failure

to

send

or

receive

cues

might

result

in

neighbouring

cells

adopting

a

randomised

polarity

Control

clones

lacking

only

fz

activity

-LRB-

marked

by

lack

of

lacZ

expression

-RRB-

show

trichomes

pointing

towards

the

clone

-LRB-

arrow

Fig.

-RRB-

whereas

stbm/Vang

clones

-LRB-

marked

by

lack

of

lacZ

-RRB-

show

trichomes

pointing

away

-LRB-

arrow

Fig.

-RRB-

We

then

generated

double

mutant

stbm/Vang

fz

clones

using

alleles

of

both

fz

and

stbm/Vang

and

an

Arm-fz-EGFP

transgene

-LRB-

which

rescues

fz

activity

see

Strutt

-RRB-

located

on

the

same

chromosome

arm

as

stbm/Vang

This

resulted

in

genetically

mosaic

wings

containing

clones

of

cells

of

the

genotype

juxtaposed

to

twinspot

tissue

of

the

genotype

Arm-fz-EGFP/Arm-fz-EGFP

or

heterozygous

tissue

of

the

genotype

-LRB-

see

Materials

and

methods

-RRB-

Such

clones

of

stbm/Vang

fz

cells

-LRB-

marked

by

lack

of

Fz-EGFP

green

-RRB-

show

negligible

effects

on

the

polarity

of

trichomes

in

neighbouring

cells

-LRB-

Fig.

trichomes

visualised

by

labelling

for

Actin

magenta

-RRB-

This

result

fits

the

hypothesis

that

bidirectional

interactions

occur

between

Fz

and

Stbm/Vang

expressing

cells

and

that

lack

of

communication

with

cells

within

a

clone

leads

to

neighbouring

cells

adopting

a

wild-type

polarity

Interestingly

within

the

double

mutant

clones

trichome

polarity

is

also

relatively

unperturbed

-LRB-

Fig.

-RRB-

This

is

in

contrast

to

single

mutant

fz

and

stbm/Vang

clones

where

trichomes

of

a

sufficient

age

adopt

polarities

consistent

with

those

shown

by

trichomes

outside

the

clone

-LRB-

e.g.

Fig.

-RRB-

We

do

not

fully

understand

this

phenomenon

however

it

is

well-established

that

trichomes

within

fz

and

stbm/Vang

tissue

largely

emerge

in

the

cell

centre

without

obvious

polarity

-LRB-

Wong

and

Adler

Taylor

et

al.

-RRB-

We

surmise

that

such

apolar

trichomes

subsequently

align

themselves

with

the

strongly

polarised

trichomes

in

the

wild-type

tissue

surrounding

the

clone

possibly

as

a

result

of

cytoskeletal

interactions

between

adjacent

cells

We

also

examined

the

distribution

of

the

core

polarity

protein

Fmi/Stan

on

the

boundaries

of

clones

of

cells

singly

or

doubly

mutant

for

fz

and

stbm/Vang

It

has

previously

been

shown

that

Fmi/Stan

strongly

localises

to

the

boundaries

between

fz

and

fz

tissue

and

stbm/Vang

and

stbm/Vang

tissue

-LRB-

arrowheads

Figs.

B

Usui

et

al.

Bastock

et

al.

-RRB-

Although

not

formally

proven

it

is

widely

thought

that

such

localised

protein

localisation

might

mediate

cell

cell

communication

-LRB-

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

Consistent

with

this

view

there

is

no

strong

localisation

of

Fmi/Stan

on

the

boundaries

of

stbm/Vang

fz

double

clones

-LRB-

Fig.

-RRB-

So

far

our

results

suggest

that

Fz

in

one

cell

and

Stbm/Vang

in

the

adjacent

cell

is

necessary

for

cell

cell

communication

and

polarisation

of

trichomes

We

next

investigated

whether

Fz

and

Stbm/Vang

in

adjacent

cells

were

sufficient

for

this

process

To

do

this

we

examined

the

effect

of

juxtaposing

cells

that

lack

fz

activity

to

cells

that

lack

stbm/Vang

activity

This

was

achieved

by

generating

clones

homozygous

for

the

genotype

Arm-Fz-EGFP

juxtaposed

to

twinspots

of

the

genotype

Arm-lacZ

-LRB-

see

Materials

and

methods

-RRB-

such

that

cells

lacking

stbm/Vang

activity

also

lacked

lacZ

expression

whereas

cells

lacking

both

fz

activity

and

the

rescuing

Arm-fz-EGFP

transgene

exhibited

high

levels

of

lacZ

expression

On

the

boundaries

where

stbm/Vang

tissue

is

juxtaposed

to

fz

tissue

we

observe

strong

Fmi/Stan

localisation

-LRB-

Fig.

arrowheads

in

Fig.

-RRB-

resembling

that

seen

on

the

edges

of

fz

or

stbm/Vang

clones

-LRB-

Figs.

B

-RRB-

Notably

at

the

edges

of

fz

and

stbm/Vang

clones

localised

Fmi/Stan

is

associated

with

production

of

polarised

trichomes

-LRB-

Figs.

B

-RRB-

apparently

as

a

result

of

assembly

of

a

polarised

asymmetric

core

polarity

protein

complex

with

Fz

on

one

side

of

the

cell

cell

boundary

and

Stbm/Vang

on

the

other

side

-LRB-

Fig.

-RRB-

We

also

observe

polarised

trichomes

produced

at

the

site

of

Fmi/Stan

localisation

on

boundaries

between

stbm/Vang

and

fz

tissue

which

point

towards

the

fz

tissue

-LRB-

Figs.

D

-RRB-

Taken

together

the

localisation

of

Fmi/Stan

and

the

production

of

polarised

trichomes

suggest

that

a

functional

core

polarity

protein

complex

assembles

on

the

boundaries

between

stbm/Vang

and

fz

tissue

and

that

this

complex

is

sufficient

to

specify

polarised

trichome

formation

We

note

that

within

the

stbm/Vang

and

fz

mutant

tissue

there

is

no

assembly

of

asymmetric

complexes

and

trichome

placement

is

unpredictable

-LRB-

Figs.

D

-RRB-

as

expected

from

previous

work

-LRB-

Wong

and

Adler

Taylor

et

al.

-RRB-

We

next

analysed

the

role

of

Fmi/Stan

in

cell

cell

communication

of

polarity

cues

in

the

wing

Unlike

fz

or

stbm/Vang

clones

clones

of

cells

lacking

fmi/stan

activity

do

not

strongly

affect

the

polarity

of

neighbouring

cells

-LRB-

Fig.

Chae

et

al.

Usui

et

al.

-RRB-

This

could

be

interpreted

to

suggest

that

fmi/stan

is

not

required

for

cell

cell

communication

and

coordination

of

cell

polarity

However

the

Fmi/Stan

protein

is

thought

to

act

as

a

homophilic

cell

adhesion

molecule

-LRB-

Usui

et

al.

-RRB-

and

so

alternatively

Fmi/Stan

may

be

required

in

both

sending

and

receiving

cells

for

coordination

of

cell

polarity

and

loss

of

fmi/stan

blocks

cell

cell

communication

Support

for

this

second

view

comes

from

experiments

in

the

abdomen

where

cells

overexpressing

Fz

or

Stbm/Vang

are

unable

to

repolarise

their

neighbours

if

they

also

lack

fmi/stan

activity

-LRB-

Lawrence

et

al.

-RRB-

We

generated

clones

of

cells

double

mutant

for

either

fz

and

fmi/stan

or

for

stbm/Vang

and

fmi/stan

Both

showed

a

phenotype

typical

of

single

mutant

fmi/stan

clones

-LRB-

Figs.

G

-RRB-

indicating

that

Fmi/Stan

is

required

in

both

Fz

and

Stbm/Vang

expressing

cells

for

cell

cell

communication

and

thus

by

extension

is

required

on

both

sides

of

the

cell

cell

boundary

Thus

for

the

fz-dependent

process

of

cell

cell

communication

that

is

thought

to

locally

coordinate

cell

polarity

we

have

demonstrated

that

Fz

and

Stbm/Vang

are

required

in

opposite

cells

and

that

Fmi/Stan

is

required

in

both

cells

This

spatial

arrangement

is

as

seen

in

the

asymmetric

complex

that

assembles

at

the

site

of

trichome

initiation

-LRB-

Fig.

-RRB-

and

supports

models

in

which

a

subset

of

this

complex

is

also

involved

in

intercellular

communication

dsh

and

pk

are

not

required

for

intercellular

communication

In

the

late

asymmetric

complex

Dsh

associates

with

Fz

at

distal

cell

edges

whereas

Pk

localises

with

Stbm/Vang

at

proximal

cell

edges

Hence

it

is

possible

that

the

association

of

Dsh

with

Fz

is

essential

for

signalling

to

Stbm/Vang

in

the

adjacent

cell

and

similarly

that

Pk

association

with

Stbm/Vang

is

required

for

signalling

to

Fz

in

the

adjacent

cell

However

interactions

have

also

been

reported

between

Dsh

and

Pk

and

between

Dsh

and

Stbm/Vang

-LRB-

Tree

et

al.

Bastock

et

al.

Jenny

et

al.

-RRB-

and

during

an

early

phase

of

cell

cell

communication

these

interactions

might

also

be

important

Clones

of

cell

mutant

for

dsh

alone

do

not

strongly

affect

the

polarity

of

neighbouring

cells

-LRB-

Fig.

Theisen

et

al.

-RRB-

in

this

respect

resembling

fmi/stan

clones

Thus

like

fmi/stan

it

is

possible

that

dsh

might

be

required

on

both

sides

of

cell

cell

boundaries

for

communication

to

occur

consistent

with

its

known

physical

associations

with

both

Fz

and

Stbm/Vang

However

we

found

that

both

dsh

fz

and

dsh

stbm/Vang

double

mutant

clones

showed

non-autonomous

effects

on

the

polarity

of

neighbouring

cells

typical

of

either

single

mutant

fz

or

stbm/Vang

clones

respectively

-LRB-

arrows

Figs.

C

-RRB-

with

normal

accumulation

of

Fmi/Stan

at

clone

boundaries

-LRB-

arrowheads

Fig.

-RRB-

These

results

demonstrate

that

dsh

activity

is

not

required

for

Fz-Stbm/Vang-dependent

intercellular

communication

to

occur

in

either

Fz-expressing

or

Stbm/Vang-expressing

cells

We

carried

out

similar

experiments

using

a

allele

of

pkpk-sple

Single

mutant

clones

of

pk

also

do

not

significantly

affect

the

polarity

of

neighbouring

cells

-LRB-

Fig.

Gubb

et

al.

-RRB-

However

pk-sple

fz

and

pk-sple

stbm/Vang

double

mutant

clones

still

show

the

typical

non-autonomous

effects

of

fz

or

stbm/Vang

clones

respectively

-LRB-

arrows

Figs.

F

-RRB-

and

accumulation

of

Fmi/Stan

at

clone

boundaries

-LRB-

arrowheads

Fig.

-RRB-

We

conclude

that

pk

activity

is

also

not

essentially

required

for

Fz-Stbm/Vang-dependent

intercellular

communication

The

range

of

non-autonomous

alterations

in

cell

polarity

around

fz

and

stbm/Vang

clones

is

generally

up

to

cell

diameters

however

it

varies

with

clone

size

shape

and

position

-LRB-

Vinson

and

Adler

Taylor

et

al.

Adler

et

al.

-RRB-

We

observed

similar

ranges

of

non-autonomy

for

the

double

mutant

clones

generated

with

dsh

and

pk

alleles

suggesting

that

the

strength

of

intercellular

signalling

remained

in

the

normal

range

Analysis

of

dgo

function

It

has

been

recently

reported

that

during

planar

polarity

patterning

of

the

Drosophila

eye

disc

the

core

polarity

gene

dgo

acts

redundantly

with

stbm/Vang

and

pk

-LRB-

Das

et

al.

-RRB-

If

such

a

situation

also

pertained

in

the

wing

we

reasoned

that

this

might

mask

specific

roles

of

either

pk

or

dgo

in

intercellular

signalling

However

we

find

that

stbm/Vang

dgo

clones

still

exhibit

the

proximal

non-autonomy

typical

of

stbm/Vang

single

clones

-LRB-

arrow

Fig.

-RRB-

and

pk

dgo

clones

behave

like

pk

single

clones

showing

no

non-autonomous

effect

on

trichome

polarity

-LRB-

Fig.

-RRB-

In

an

attempt

to

test

the

hypothesis

of

redundant

functions

of

core

polarity

proteins

as

rigorously

as

possible

we

generated

clones

of

cells

triply

mutant

for

pk

dgo

and

fz

These

also

behaved

like

single

mutant

fz

clones

showing

typical

non-autonomous

effects

on

trichome

polarity

-LRB-

arrow

Fig.

-RRB-

In

the

eye

dgo

has

been

particularly

implicated

in

cooperating

with

pk

and

stbm/Vang

to

maintain

the

junctional

localisation

of

Fmi/Stan

-LRB-

Das

et

al.

-RRB-

Interestingly

in

the

pupal

wing

Fmi/Stan

remains

junctional

in

stbm/Vang

dgo

clones

-LRB-

Fig.

-RRB-

although

proximodistal

asymmetric

localisation

is

lost

as

previously

reported

for

stbm/Vang

clones

-LRB-

Bastock

et

al.

-RRB-

The

same

is

true

of

Fmi/Stan

localisation

in

pk

dgo

clones

-LRB-

Fig.

-RRB-

We

also

find

that

contrary

to

the

reported

situation

in

the

eye

Stbm/Vang

apicolateral

localisation

is

maintained

in

pk

dgo

clones

-LRB-

Fig.

-RRB-

Polarity

defects

propagate

through

tissue

mutant

for

pk

pk

dgo

and

dsh

to

different

degrees

Mosaic

experiments

so

far

described

have

analysed

the

ability

of

clones

of

cells

lacking

the

function

of

one

or

more

core

polarity

genes

to

influence

the

polarity

of

neighbouring

cells

via

intercellular

signalling

However

for

polarity

defects

to

propagate

away

from

such

clones

intracellular

signalling

is

required

across

the

axes

of

individual

cells

in

addition

to

intercellular

signalling

between

cells

The

nature

of

intracellular

signalling

is

poorly

understood

One

recent

model

proposes

that

it

depends

upon

detection

of

levels

of

intracellular

fz

activity

by

transmembrane

receptors

on

different

sides

of

the

cell

-LRB-

Lawrence

et

al.

-RRB-

whereas

others

suggest

that

it

relies

on

asymmetric

assembly

of

protein

complexes

on

one

cell

edge

in

response

to

the

presence

of

an

asymmetric

complex

at

the

opposite

cell

edge

-LRB-

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

Although

dsh

pk

and

dgo

do

not

have

essential

functions

in

intercellular

signalling

they

might

nevertheless

be

required

for

intracellular

communication

However

in

this

context

it

is

interesting

to

note

that

polarity

defects

can

still

propagate

around

fz

clones

in

abdomens

wholly

mutant

for

a

allele

of

pk

-LRB-

Lawrence

et

al.

-RRB-

and

in

wings

mutant

for

the

mutation

which

mutates

one

of

the

pk

isoforms

-LRB-

Adler

et

al.

-RRB-

Taken

at

face

value

these

data

suggest

that

pk

may

not

be

essentially

required

for

either

intercellular

or

intracellular

signalling

To

investigate

this

further

we

generated

clones

of

cells

with

altered

fz

or

stbm/Vang

activity

in

wings

wholly

mutant

for

the

function

of

other

core

polarity

genes

As

it

simplified

the

generation

of

the

appropriate

fly

strains

in

these

experiments

we

generated

clones

of

cells

with

increased

Fz

activity

-LRB-

which

cause

neighbouring

trichomes

to

point

away

from

the

clone

see

e.g.

Strutt

-RRB-

or

Stbm/Vang

activity

-LRB-

which

cause

trichomes

to

point

towards

the

clone

Amonlirdviman

et

al.

-RRB-

For

control

experiments

we

analysed

wings

entirely

mutant

for

fz

or

stbm/Vang

activity

These

factors

are

required

both

for

intercellular

communication

and

trichome

placement

at

the

cell

edge

so

overexpression

clones

in

these

backgrounds

are

not

expected

to

alter

the

polarity

of

neighbouring

cells

-LRB-

e.g.

Taylor

et

al.

-RRB-

Consistent

with

this

clones

of

cells

that

overexpress

Stbm/Vang

can

not

alter

trichome

polarity

in

fz

wings

-LRB-

Fig.

-RRB-

and

clones

of

cells

overexpressing

Fz

can

not

alter

trichome

polarity

in

stbm/Vang

wings

-LRB-

Fig.

-RRB-

However

if

Fz

is

overexpressed

in

clones

of

cells

in

wings

wholly

mutant

for

a

allele

of

pk

we

observed

a

strong

effect

on

trichome

polarity

in

neighbouring

cells

-LRB-

Fig.

-RRB-

which

extends

cells

-LRB-

average

n

-RRB-

from

the

clone

boundary

similar

to

the

effect

seen

in

wild-type

wings

and

in

agreement

with

previous

observations

-LRB-

Adler

et

al.

Lawrence

et

al.

-RRB-

Interestingly

in

wings

double

mutant

for

pk

and

dgo

clones

of

cells

overexpressing

Fz

also

affect

the

polarity

of

neighbouring

cells

-LRB-

Fig.

-RRB-

but

the

effect

only

extends

for

cells

-LRB-

average

n

-RRB-

Hence

although

patterning

is

essentially

normal

in

dgo

wings

and

polarity

defects

propagate

a

normal

distance

though

pk

tissue

when

both

factors

are

absent

the

propagation

of

polarity

defects

is

substantially

reduced

revealing

an

unexpected

redundancy

between

these

factors

for

this

process

We

also

investigated

the

effect

of

overexpressing

Fz

in

clones

of

cells

in

wings

mutant

for

dsh

To

circumvent

the

lethality

of

dsh

mutations

we

used

the

point

mutation

which

affects

only

planar

polarity

functions

We

again

observed

an

effect

of

the

clones

on

the

polarity

of

neighbouring

cells

-LRB-

Fig.

-RRB-

however

in

this

case

polarity

defects

only

propagated

at

most

cells

from

the

clone

-LRB-

average

n

-RRB-

Finally

we

tested

propagation

through

cells

mutant

for

a

allele

of

dsh

Non-autonomous

polarity

defects

were

induced

by

overexpressing

Fz-GFP

in

the

patched

expression

domain

at

the

anteroposterior

compartment

boundary

-LRB-

Adler

et

al.

-RRB-

in

wings

containing

clones

-LRB-

Fig.

-RRB-

Within

the

clones

we

observed

trichomes

emerging

largely

in

the

cell

centre

as

previously

reported

-LRB-

Wong

and

Adler

-RRB-

and

were

unable

to

detect

propagation

of

polarity

defects

Asymmetry

of

polarity

protein

activity

exists

in

the

absence

of

asymmetry

in

distribution

Recent

models

have

suggested

that

asymmetric

localisation

might

be

essential

for

polarity

propagation

-LRB-

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

This

is

at

variance

with

observations

of

propagation

of

polarity

defects

in

pk

or

backgrounds

-LRB-

Figs.

E

and

Adler

et

al.

Lawrence

et

al.

-RRB-

in

which

asymmetry

is

not

observed

-LRB-

Figs.

B

and

e.g.

Axelrod

Strutt

Usui

et

al.

-RRB-

However

it

is

possible

that

weak

asymmetry

exists

in

these

backgrounds

which

is

difficult

to

detect

We

investigated

this

more

closely

by

generating

clones

of

cells

expressing

Fz-EYFP

In

general

a

subtle

increase

or

decrease

in

Fz-EYFP

at

the

edge

of

one

cell

might

be

masked

by

Fz-EYFP

localisation

on

the

adjacent

boundary

of

a

neighbouring

cell

-LRB-

as

it

is

not

possible

to

distinguish

between

localisation

at

adjacent

cell

boundaries

by

visible

light

microscopy

-RRB-

However

by

looking

at

Fz-EYFP

distribution

on

the

edges

of

clones

it

should

be

easier

to

discern

subtle

asymmetry

of

proximodistal

localisation

-LRB-

see

Strutt

-RRB-

Hence

we

examined

the

distribution

of

Fz-EYFP

expressed

in

clones

in

pk

and

backgrounds

but

still

were

unable

to

observe

any

evidence

of

asymmetric

localisation

-LRB-

Figs.

B

-RRB-

Notwithstanding

our

failure

to

find

asymmetry

of

distribution

of

polarity

proteins

in

pk

and

wings

we

nevertheless

suppose

that

there

must

be

asymmetry

of

activity

in

order

for

propagation

of

polarity

and

asymmetric

trichome

placement

to

occur

Interestingly

in

clones

of

cells

lacking

dgo

or

pk

activity

trichome

formation

occurs

both

at

the

same

time

as

in

adjacent

non-mutant

tissue

and

at

the

distal

cell

edge

-LRB-

Figs.

D

-RRB-

Hence

in

these

backgrounds

there

is

clearly

an

asymmetric

signal

for

trichome

formation

In

contrast

in

clones

of

cells

doubly

mutant

for

dgo

and

pk

trichome

formation

in

often

seen

to

be

delayed

and

when

it

does

occur

is

often

seen

in

the

cell

centre

-LRB-

Fig.

-RRB-

indicative

of

loss

of

the

asymmetric

trichome

placement

cue

The

phenotype

in

pk

dgo

clones

is

similar

to

that

seen

in

tissue

mutant

for

dsh

fz

or

stbm/Vang

-LRB-

Fig.

Wong

and

Adler

Taylor

et

al.

-RRB-

Hence

consistent

with

the

differing

effects

of

dsh

pk

and

dgo

on

propagation

of

polarity

defects

we

see

a

similar

progression

of

effects

on

the

asymmetric

placement

of

trichomes

at

the

cell

edge

suggesting

that

these

two

processes

are

linked

Discussion

As

described

in

the

Introduction

it

is

possible

to

define

a

core

group

of

polarity

proteins

but

this

does

not

imply

that

all

components

of

the

core

make

equal

contributions

to

planar

polarity

patterning

In

this

work

we

have

attempted

to

systematically

analyse

the

contributions

of

the

core

proteins

to

the

processes

of

coupling

to

the

global

cue

local

coordination

of

polarity

and

asymmetric

trichome

placement

Our

key

findings

are

as

follows

-LRB-

i

-RRB-

The

transmembrane

proteins

Fz

Stbm/Vang

and

Fmi/Stan

have

a

common

early

function

during

planar

polarity

patterning

in

the

wing

and

eye

with

the

cytoplasmic

factors

Dsh

and

Pk

playing

only

later

roles

-LRB-

ii

-RRB-

The

transmembrane

core

of

Fz

Fmi/Stan

and

Stbm/Vang

is

absolutely

required

for

intercellular

communication

We

demonstrate

that

the

asymmetric

relationship

of

these

proteins

seen

at

the

time

of

trichome

placement

with

Fmi/Stan

in

both

communicating

cells

and

Fz

in

one

cell

juxtaposed

to

Stbm/Vang

in

the

adjacent

cell

is

also

necessary

and

sufficient

for

such

intercellular

communication

In

addition

we

provide

evidence

that

information

passes

both

from

Fz

to

Stbm/Vang

expressing

cells

and

vice

versa

-LRB-

iii

-RRB-

Intercellular

communication

does

not

require

Dsh

or

Pk

in

either

Fz

or

Stbm/Vang

expressing

cells

and

Dgo

also

does

not

play

redundant

roles

with

Pk

in

intercellular

signalling

-LRB-

iv

-RRB-

Pk

and

Dgo

act

redundantly

in

propagation

of

polarity

from

cell

to

cell

most

likely

by

promoting

intracellular

communication

Dsh

plays

a

prominent

role

in

such

propagation

greater

than

that

of

both

Pk

and

Dgo

We

speculate

that

the

intracellular

communication

required

for

such

polarity

propagation

is

dependent

on

establishing

intracellular

asymmetries

of

protein

activity

-LRB-

v

-RRB-

Although

even

subtle

asymmetry

of

Fz

localisation

is

not

apparent

in

pk

tissue

not

only

does

polarity

propagate

between

cells

but

trichome

placement

also

occurs

at

the

normal

time

and

place

Hence

asymmetry

of

polarity

protein

activity

exists

in

the

absence

of

detectable

asymmetry

of

localisation

-LRB-

vi

-RRB-

Neither

Pk

nor

Dgo

are

directly

required

for

determining

the

site

of

trichome

placement

Coupling

to

the

global

polarity

cue

As

noted

in

the

Introduction

one

of

the

putative

functions

of

the

core

polarity

proteins

is

to

couple

to

long-range

polarity

patterning

cues

It

has

been

suggested

that

these

cues

are

provided

by

fj/ds/ft

-LRB-

Ma

et

al.

Yang

et

al.

-RRB-

but

little

is

understood

regarding

the

molecular

mechanism

of

any

such

coupling

We

suppose

that

fz

stbm/Vang

and

fmi/stan

are

involved

as

they

show

the

earliest

requirement

Comparison

with

the

temporal

requirements

of

ds

-LRB-

Matakatsu

and

Blair

-RRB-

argues

against

a

role

for

pk

and

probably

dsh

-LRB-

with

the

caveat

that

we

were

unable

to

analyse

a

allele

-RRB-

Interestingly

it

has

been

argued

that

during

abdomen

patterning

pk

may

play

a

particular

role

in

rectifying

the

global

signal

in

different

compartments

-LRB-

Lawrence

et

al.

-RRB-

In

the

wing

such

a

function

is

not

necessary

possibly

explaining

why

we

do

not

find

a

corresponding

early

role

for

pk

Local

coordination

of

polarity

A

better

understood

and

major

function

of

Fz

and

the

core

polarity

proteins

is

the

local

coordination

of

cell

polarity

All

recent

models

for

this

coordination

have

proposed

a

role

for

cell

cell

contact

mediated

signalling

as

opposed

to

schemes

requiring

the

secretion

of

a

diffusible

ligand

A

key

feature

of

such

models

is

that

they

require

both

intercellular

communication

to

pass

polarity

cues

between

adjacent

cells

and

intracellular

communication

to

pass

information

across

the

axes

of

individual

cells

Experimental

support

for

cell

cell

contact

mediated

signalling

has

been

provided

by

experiments

in

the

abdomen

showing

that

the

atypical

cadherin

Fmi/Stan

is

required

in

both

signal

sending

and

receiving

cells

suggesting

that

signals

pass

between

Fmi/Stan

homodimers

-LRB-

Lawrence

et

al.

-RRB-

Theoretical

evidence

has

also

been

provided

by

a

number

of

mathematical

models

that

have

confirmed

the

feasibility

of

locally

coordinating

polarity

via

assembly

of

asymmetric

junctional

complexes

containing

Fz

in

one

cell

and

Stbm/Vang

in

the

adjacent

cell

-LRB-

Amonlirdviman

et

al.

Le

Garrec

et

al.

-RRB-

Our

results

rigorously

demonstrate

that

in

the

wing

intercellular

signalling

events

that

locally

coordinate

polarity

require

Fmi/Stan

in

both

communicating

cells

and

Fz

in

one

cell

and

Stbm/Vang

in

the

other

This

supports

models

in

which

the

asymmetric

junctional

distributions

that

are

observed

by

immunofluorescence

are

required

for

intercellular

signalling

-LRB-

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

However

it

should

be

noted

that

although

signalling

may

require

the

assembly

of

complexes

with

Fz

in

one

cell

adjacent

to

Stbm/Vang

in

the

next

this

does

not

necessarily

imply

that

detectable

asymmetric

subcellular

distribution

of

proteins

within

cells

is

necessary

Indeed

the

persistence

of

signalling

in

pk

and

backgrounds

where

subcellullar

asymmetry

of

the

core

polarity

proteins

is

not

observed

argues

against

this

being

essential

In

addition

our

data

raise

the

possibility

of

bidirectional

cell

cell

communication

via

Fz-Stbm/Vang

and

are

inconsistent

with

a

monodirectional

signal

as

proposed

to

occur

in

the

abdomen

-LRB-

Lawrence

et

al.

-RRB-

It

is

also

evident

from

our

results

that

intercellular

signalling

does

not

require

association

of

Dsh

Pk

or

Dgo

with

Fz

or

Stbm/Vang

Indeed

fz

or

stbm/Vang

clones

that

also

lack

any

of

these

factors

are

not

obviously

impaired

in

their

ability

to

alter

the

polarity

of

neighbouring

cells

What

then

are

the

roles

of

Dsh

Pk

and

Dgo

We

show

that

propagation

of

polarity

defects

away

from

a

clone

is

reduced

in

dsh

and

pk

dgo

tissue

indicating

that

they

are

required

for

local

relay

of

polarity

cues

Hence

a

likely

role

would

be

in

the

intracellular

signalling

required

to

pass

polarity

cues

across

the

axes

of

cells

Previous

experiments

in

which

polarity

was

seen

to

propagate

normally

in

a

pk

background

-LRB-

Adler

et

al.

Lawrence

et

al.

-RRB-

argued

against

such

a

function

for

pk

which

is

only

revealed

when

dgo

function

is

also

absent

One

proposed

mechanism

for

intracellular

signalling

is

that

each

cell

acquires

a

particular

level

of

Fz

activity

which

is

communicated

by

intercellular

signalling

to

all

surrounding

cells

-LRB-

Lawrence

et

al.

-RRB-

In

this

case

roles

for

Dsh

Pk

and

Dgo

in

modulating

intracellular

levels

of

Fz

activity

could

be

envisaged

However

the

majority

of

models

suggest

roles

for

Dsh

and

Pk

in

intracellular

feedback

loops

that

amplify

differences

in

the

asymmetric

localisation

of

the

core

polarity

protein

complexes

within

cells

-LRB-

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

For

instance

it

has

been

proposed

that

association

of

Dsh

with

Fz

might

be

antagonised

by

high

local

concentration

of

Stbm/Vang-Pk

-LRB-

Amonlirdviman

et

al.

-RRB-

or

alternatively

that

Fz-Dsh

antagonise

Stbm/Vang-Pk

interactions

-LRB-

Le

Garrec

et

al.

-RRB-

In

either

case

if

asymmetric

complexes

containing

Fz

Fmi/Stan

and

Stbm/Vang

were

somehow

stabilised

by

addition

of

Dsh

Pk

and/or

Dgo

to

the

complex

then

such

antagonistic

interactions

would

provide

feedback

that

would

amplify

asymmetries

of

protein

localisation

across

the

axes

of

individual

cells

Notably

our

experimental

results

suggest

that

if

such

feedback

is

occurring

then

the

relative

importance

of

the

cytoplasmic

factors

in

stabilising

complex

formation

follows

the

hierarchy

Dsh

Pk

Dgo

Such

a

scheme

would

also

explain

the

redundant

functions

of

pk

and

dgo

even

though

these

factors

act

at

opposite

cell

edges

as

Pk

on

one

side

of

a

cell

cell

boundary

could

bind

to

and

stabilise

a

complex

that

was

also

being

stabilised

by

Dgo

binding

on

the

opposite

side

of

the

cell

cell

boundary

Simultaneous

loss

of

both

Pk

and

Dgo

would

have

a

greater

destabilising

effect

than

loss

of

either

factor

alone

However

models

that

depend

on

amplification

of

differences

in

asymmetric

subcellular

protein

distribution

have

to

be

reconciled

with

the

failure

to

observe

protein

asymmetries

in

pk

or

tissue

through

which

polarity

can

still

propagate

Possibly

in

these

backgrounds

there

are

subtle

asymmetries

which

can

not

be

observed

notably

at

least

one

recent

model

predicts

such

subtle

asymmetry

in

pk

clones

-LRB-

Amonlirdviman

et

al.

-RRB-

But

another

explanation

is

that

receptor

proteins

such

as

Fz

may

be

uniformly

distributed

but

nevertheless

exhibit

differential

signalling

activity

across

the

axes

of

cells

Despite

the

apparently

non-essential

role

of

protein

asymmetry

either

in

polarity

propagation

over

short

distances

or

in

trichome

placement

it

nevertheless

seems

likely

that

it

is

an

active

mechanism

in

ensuring

robust

coordination

of

polarity

and

correct

trichome

placement

over

the

whole

wing

-LRB-

Ma

et

al.

Amonlirdviman

et

al.

-RRB-

as

otherwise

the

failure

of

long-range

coordination

of

polarity

in

pk

wings

can

not

be

explained

Interestingly

it

has

recently

been

reported

that

asymmetry

is

present

from

as

early

as

the

third

instar

stage

of

development

but

is

subsequently

lost

during

junctional

remodelling

in

pupal

stages

-LRB-

Classen

et

al.

-RRB-

suggesting

that

such

asymmetry

could

be

playing

a

role

from

much

earlier

in

development

than

previously

suspected

Specification

of

the

site

of

polarised

trichome

production

The

precise

mechanism

by

which

asymmetric

trichomes

are

generated

remains

unknown

although

there

appears

to

be

a

role

for

asymmetric

subcellular

activities

of

polarity

effector

proteins

such

as

Inturned

-LRB-

Adler

et

al.

-RRB-

As

asymmetric

trichomes

can

be

generated

in

the

absence

of

Pk

or

Dgo

it

seems

unlikely

that

either

of

these

proteins

interacts

directly

with

the

trichome

placement

machinery

however

all

of

the

other

core

polarity

proteins

are

candidates

for

such

a

role

Concluding

remarks

In

conclusion

we

note

that

there

has

recently

been

great

interest

in

attempting

to

mathematically

model

the

processes

underlying

propagation

of

planar

polarity

between

cells

-LRB-

e.g.

Lawrence

et

al.

Amonlirdviman

et

al.

Klein

and

Mlodzik

Le

Garrec

et

al.

-RRB-

Although

the

presented

models

have

been

very

successful

at

reproducing

known

phenomena

they

have

nevertheless

been

based

on

limited

experimental

data

This

work

both

provides

support

for

some

of

the

assumptions

of

such

models

for

instance

by

directly

testing

the

central

role

of

core

transmembrane

proteins

such

as

Fz

Fmi/Stan

and

Stbm/Vang

in

intercellullar

signalling

but

also

provides

challenges

for

instance

by

demonstrating

the

propagation

of

polarity

in

the

absence

of

visible

protein

asymmetry

planar polarity
pcp
drosophila
frizzled
strabismus
flamingo
dishevelled
prickle
diego