Technological advancements
GFP as a tool to analyze the organization,dynamics and function
of nuclei and microtubules in Neurospora crassa
Michael Freitag a,*,1,Patrick C.Hickey b,c,1,Namboori B.Raju d,1,
Eric U.Selker a ,Nick D.Read b
a汪沛英
Institute of Molecular Biology and Department of Biology,University of Oregon,Eugene,OR 97403,USA
b
Fungal Cell Biology Group,Institute of Cell Biology,Rutherford Building,University of Edinburgh,Edinburgh EH93JH,UK
c
LUX Biotechnology,Edinburgh Technology Transfer Centre,Alrick Building,Edinburgh EH93JL,UK
d
修伟良Department of Biological Sciences,Stanford University,Stanford,CA 94305,USA
Received 13April 2004;accepted 28June 2004
Abstract
We report the construction of a versatile GFP expression plasmid and demonstrate its utility in Neurospora crassa .To visualize nuclei and microtubules,we generated carboxy-terminal fusions of sgfp to Neurospora histone H1(hH1)and b -tubulin (Bml ).Strong expression of GFP fusion proteins was achieved with the inducible Neurospora ccg-1promoter.Nuclear and microtubule organiza-tion and dynamics were observed in live vegetative hyphae,developing asci,and ascospores by conventional and confocal laser scan-ning fluorescence microscopy.Observations of GFP fusion proteins in live cells largely confirmed previous results obtained by examination of fixed cells with various microscopic techniques.H1-GFP revealed dynamic nuclear shapes.Microtubules were mostly aligned parallel to the growth axis in apical compartments but more randomly arranged in sub-apical compartments.Time-lapse imaging of b -tubulin-GFP in germinating macroconidia revealed polymerization and depolymerization of microtubules.In heterozygous crosses,H1-GFP and 灵溪一中
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b -tubulin-GFP expression was silenced,presumably by meiotic silencing.H1-GFP was trans-lated in the vicinity of hH1+-sgfp +nuclei in the common cytoplasm of giant Banana ascospores,but it diffused into all nuclei,another illustration of the utility of GFP fusion proteins.Ó2004Elsevier Inc.All rights reserved.
Index Descriptors:Green fluorescent protein;b -Tubulin;Microtubules;Histone H1;Meiotic silencing by unpaired DNA;Ascus development;
Confocal microscopy
1.Introduction
Fluorescent proteins are powerful tools that have rev-olutionized cell biology over the last decade,particularly in studies of living cells.Since the initial report of heter-ologous expression of green fluorescent protein (GFP)from the jellyfish Aequorea victoria in Escherichia coli and Caenorhabditis elegans (Chalfie et al.,1994),GFP and other fluorescent proteins have been adapted (Tsien,
1998;Zimmer,2002)for use in many experimental sys-tems,including numerous species of fungi (Bou
rett et al.,2002;Lorang et al.,2001).Genetically encoded fluorescent proteins are used in a wide variety of appli-cations in live-cell imaging,including measurement of dynamic gene expression,localization of DNA and pro-teins,interactions between proteins,enzyme activation,calcium and cAMP signalling,and pH homeostasis (e.g.,Brandizzi et al.,2002;Czymmek et al.,2002;Heun et al.,2001;Lippincott-Schwartz et al.,2003;Miyawaki,2003;Robinett et al.,2001;Zhang et al.,2002).
Although many attempts to express versions of GFP under the control of constitutive or inducible fungal
1087-1845/$-see front matter Ó2004Elsevier Inc.All rights reserved.doi:10.1016/j.fgb.2004.06.008
*
Corresponding author.Fax:+15413465891.
E-mail address:freitag@molbio.uoregon.edu (M.Freitag).1
These authors contributed equally to this work.
www.elsevier/locate/yfgbi
Fungal Genetics and Biology 41(2004)
897–910
promoters at levels necessary for live-cell imaging in Neurospora crassa were unsuccessful,we achieved cyto-plasmic expression of a GFP derivative(sgfp)in Neuro-spora(Freitag et al.,2001).The sgfp gene has been used in plants(Chiu et al.,1996)and fungi(Fernandez-Aba-los et al.,1998;Fox et al.,2002).A serine to threonine substitution at position65(S65T;Heim et al.,1995)re-sults in increased brightness,and mutations to shift the codon bias closer to that found in humans(Haas et al., 1996)assa(Borkovich et al.,2004;Galagan et al.,2003)made this GFP variant an attractive ch
oice for studies in Neurospora.A mitochondrially targeted GFP fusion protein has been successfully expressed from a strong Neurospora promoter(Fuchs et al., 2002),but this construct is not easily adaptable for gen-eral use.For the present study,we aimed to build a ver-satile‘‘cassette’’plasmid,in which Neurospora promoters and genes,as well asfluorescent proteins can be easily exchanged and integrated at a precise loca-tion in the genome.Thus,ourfirst aim was to develop methodology to achieve strong expression of GFP fu-sion proteins assa by constructing translational fusions of assa ccg-1(formerly grg-1)promoter (McNally and Free,1988)to sgfp and either the Neuro-spora histone H1(hH1;Folco et al.,2003)or b-tubulin gene(Bml;Orbach et al.,1986).The second aim was to image nuclear and microtubule organization and dynamics in growing vegetative hyphae,developing asci, and ascospores in strains transformed with these con-structs,to confirm and extend results obtained previ-ously by traditional microscopic techniques that principally made use offixed cells.
Neurospora nuclei have been studied extensively using brightfield light microscopy(Barry,1972;Bianchi and Turian,1967;Dodge,1927;McClintock,1945;Raju, 1980,1984;Singleton,1953;Wilson and Aist,1967),fluorescence microscopy(Freitag et al.,2004;Heath et al.,2000;Lee et al.,2001;Marek et al.,2003;Minke et al.,1999a,b;Perkins et al.,1995;Raju,1982,198
6; Riquelme et al.,2002;That et al.,1988;Thompson-Coffe and Zickler,1994;Tinsley et al.,1996),and trans-mission electron microscopy(Bojko,1988;Gillies,1972, 1979;Lu,1993;Riquelme et al.,2002).Most of these studies have usedfixed cells.One study involved vid-eo-enhanced DIC light microscopy of living,unstained vegetative hyphae,and immunofluorescence and laser scanning confocal microscopy(Riquelme et al.,2002). Our recent studies employed conventionalfluorescence (Folco et al.,2003;Freitag et al.,2004;Glass et al., 2004)and confocal imaging of GFP-labeled nuclei(Fre-itag et al.,2004).Similarly,studies of microtubules in Neurospora have usedfixed cells(Bruno et al.,1996; Heath et al.,2000;Minke et al.,1999a,b;Riquelme et al.,2002;Shiu et al.,2001;That et al.,1988;Thomp-son-Coffe et al.,1999).The present study on live cells largely confirms previous results obtained with tech-niques that are more invasive.Visualization of H1-GFP and b-tubulin-GFP in meiosis revealed silencing in heterozygous crosses,presumably by meiotic silencing by unpaired DNA(Shiu et al.,2001).Our results illus-trate the utility of GFP fusion proteins to study the cell biology of live Neurospora in real time.
2.Materials and methods
2.1.Construction of sgfp-containing plasmids
Wefirst generated a plasmid with the bright S65T ver-sion of GFP by inserting the Bam HI–Bgl II fragment of plasmid pFA6a-GFP(S65T)-kanMX6(Longtine et al., 1998)into Bam HI-digested pBM60(Margolin et al., 1997)to yield pMF255.The GFP(S65T)gene was re-placed by amplification of sgfp from pCT74(Lorang, 2001)and insertion of the Bam HI+Eco RI-digested frag-ment into pMF255.This yielded pMF267,a promoterless sgfp construct useful for generating translational fusions to genes driven by their endogenous promoters.To gener-ate over-expression constructs,the inducible Neurospora ccg-1(formerly grg-1)promoter(Pccg-1;McNally and Free,1988)and50untranslated region of the transcript di-rectly preceding the ccg-1AUG(nt738–1659;GenBank Accession No.L14464)was amplified by PCR and in-serted into Not I+Xba I-digested pMF267,yielding pMF272.To construct translational fusions to the N. crassa histone H1gene(hH1)and the b-tubulin gene (Bml),we amplified the hH1and Bml genes by PCR,di-gested with Bam HI+Pac I or Xma I+Pac I,and inserted fragments into pMF272,to yield pMF280and pMF309, respectively.All PCRs were performed with Herculase polymerase(Stratagene).Plasmids pMF272,pMF280, and pMF309are available from the Fungal Genetics Stock Center,Kansas City,MO and their GenBank Accession Nos.:pMF272(AY598428),pMF280 (AY598429),and pMF309(AY598430).
2.2.Transformation protocols and transformant selection
Neurospora transformations were performed by elec-troporation as described previously(Margolin et al., 1997).Plasmids pMF272and pMF280were targeted to the his-3locus of strain N623yielding the primary heterokaryotic prototrophic His+transformants N2261 and N2276and N2277,respectively(Folco et al., 2003).Plasmid pMF309was integrated at his-3of N2240and N2257,yielding N2509and N2505,respec-tively(see Table1).An Olympus SZX12fluorescence stereo microscope with a GFPfilter set(460–490nm excitation,505nm dichroic mirror and510–550nm emissionfilter)was used to screen for GFP+transfor-mants.Heterokaryotic transformants were crossed to obtain homokaryotic progeny of desired genotypes.
898M.Freitag et al./Fungal Genetics and Biology41(2004)897–910
All Neurospora plasmids based on , pMF272,pMF280,and pMF309)are designed for gene targeting to the his-3locus(Margolin et al.,1997).No additional selectable marker for fungal transformation is contained on these plasmids,but co-transformations with hph,a gene that confers resistance to hygromycin B(Staben et al.,1989),have been successfully performed assa(M.Freitag and E.U.Selker,data not shown)and Magnaporthe grisea(J-R.Xu,Purdue Uni-versity,personal communication).
2.3.Strains and culture conditions
Strains used in this study are listed in Table1.Strains were maintained on VogelÕs minimal medium and all manipulations were according to standard Neurospora techniques(Davis,2000).The heterokaryotic strain N2276was crossed to wild type N1to isolate mat A his-3+::hH1+-sgfp+(N2282)and mat a his-3+::hH1+-sgfp+(N2283)progeny,respectively.To obtain homo-karyotic hH1+-sgfp+strains for subsequent crosses that would not be complicated by repeat-induced point mutation(RIP),we also crossed the heterokaryotic N2277to a rid mat a his-3strain(N2257;Freitag et al.,2002)and isolated mat A his-3+::hH1+-sgfp+ (N2280)and rid mat a his-3+::hH1+-sgfp+(N2281), respectively.A homokaryotic mat A his-3+::hH1+-sgfp+ strain(N2276-1)was isolated from N2276by serial conidial isolation.Two additional mat A his-3+::hH1+-sgfp+strains(N2281-3and N2281-8)were isolated from a cross of mat A;fluffy to N2281,because N2276-1, N2280,and N2282carry a previously unknown asco-spore maturation defect.N2281,N2281-3,and N2281-8are free from the defect.Primary transformants N2505and N2509were crossed to isolate mat a rid RIP4his-3+::Pccg-1-Bml+-sgfp+(N2524,N2525)and mat A rid RIP1his-3+::Pccg-1-Bml+-sgfp+(N2526,N2527).
2.4.Fluorescent staining and preparation of cultures for live-cell imaging of vegetative hyphae
GFP-expressing strains were routinely grown on Vo-gelÕs minimal salt medium,supplemented with2%su-crose or on2%malt extract medium.Live-cell imaging involved using the‘‘inverted agar bloc
k method’’of pre-paring and staining samples(Hickey et al.,2002;Hickey and Read,2003).Hyphae were double-labeled for one hour with either25l M FM4-64(Molecular Probes,Eu-gene,OR),which stains the plasma membrane and orga-nelle membranes(Fischer-Parton et al.,2000),or10l M DASPMI(Molecular Probes,Eugene,OR),which stains mitochondria(Bereiter-Hahn,1990).Conidia from original H1-GFP+transformants were counter-stained with the DNA dye Hoechst33258to verify nu-clear localization of the fusion protein(data not shown and Freitag et al.,2004).
Table1
Neurospora crassa strains used in this study
Strain a Genotype Reference
N1(74-OR8-1)mat a FGSC#988
N150(74-OR23-IV)mat A FGSC#2489
fluffy mat A;fl(RL);scot FGSC#6682
fluffy mat a;fl(RL);scot FGSC#6683 Banana mat a Ban mei-3;inl FGSC#2990
per-1mat A csp-2;per-1FGSC#6662
N623mat A his-3FGSC#6103
N2240rid RIP1mat A his-3Freitag et al.(2002) N2257rid RIP4mat a his-3Freitag et al.(2002) N2261(mat A his-3+::Pccg-1-sgfp++mat A his-3)Folco et al.(2003) N2276,N2277(mat A his-3+::Pccg-1-hH1+-sgfp++mat A his-3)Folco et al.(2003) N2276-1mat A his-3+::Pccg-1-hH1+-sgfp+This study
N2280mat A his-3+::Pccg-1-hH1+-sgfp+This study
N2281rid RIP4mat a his-3+::Pccg-1-hH1+-sgfp+This study
N2281-3mat A his-3+::Pccg-1-hH1+-sgfp+This study
N2281-8mat A his-3+::Pccg-1-hH1+-sgfp+;flThis study
N2282mat A his-3+::Pccg-1-hH1+-sgfp+This study
N2283mat a his-3+::Pccg-1-hH1+-sgfp+This study
N2505(rid RIP4mat a his-3+::Pccg-1-Bml+-sgfp++rid RIP4mat a his-3)This study
N2509(rid RIP1mat A his-3+::Pccg-1-Bml+-sgfp++rid RIP1mat A his-3)This study
N2524,N2525rid RIP4mat a his-3+::Pccg-1-Bml+-sgfp+This study
N2526,N2527rid RIP1mat A his-3+::Pccg-1-Bml+-sgfp+This study
A novel ascospore maturation defect was identified in crosses of two independent transformants of N623to either N1or N2257(N2276,N2276-1, N2277,N2280,and N2282).The primary transformants N2276,N2277,N2505,and N2509contain both transformed and untransformed nuclei and are therefore denoted as heterokaryons.
a Selected strains are available from the Fungal Genetics Stock Center:N2240(FGSC#9014),N2257(FGSC#9015),N2261(FGSC#9516), N2281(FGSC#9517),N2281-3(FGSC#9518),N2524(FGSC#9519),and N2526(FGSC#9520).
M.Freitag et al./Fungal Genetics and Biology41(2004)897–910899
2.5.Confocal microscopy and image processing
Confocal laser scanning microscopy was performed using a Bio-Rad Radiance2100system equippe
d with an argon ion laser,and mounted on a Nikon TE300in-verted microscope.GFP and FM4-64or GFP and DAS-PMI,respectively,were imaged simultaneously by excitation with the488nm laser line andfluorescence detection at515–530nm(for GFP),and>600nm(for FM4-64or DASPMI).Oil immersion60·(N.A.1.4) or dry20·(N.A.0.75)plan apochromatic objective lenses were used for imaging.The laser intensity and la-ser scanning of individual hyphae were kept to a mini-mum to reduce dye photobleaching and phototoxic effects.The imaging parameters used produced no back-ground signal from any source other than from GFP or dyefluorescence(data not shown).Time-lapse imaging was performed at scan intervals of3–10s for periods up to15min.Confocal images were captured using Lasersharp software(version5.1;Bio-Rad)and were initially viewed using Confocal Assistant software(ver-sion4.02).These images were transferred into Paintshop Pro software(version7.0;JASC)for further processing. Much of the analysis of time-lapse sequences involved converting them into animation movies.For this pur-pose,time-lapse sequences were re-sampled to convert them from‘‘.pic’’to‘‘.avi’’files in order to be processed by Adobe Premiere software(version6.0).File compres-sion was often necessary to prevent‘‘stuttering’’play-back due to the limited data transfer ,the time taken to transfer data from hard disk to the video display hardware).Final animation movies(Supplemen-tary Movies1–3)accompany this report and can be downloaded from and in MPEG-1format.
2.6.Crosses for conventionalfluorescence microscopy and imaging of asci and ascospores
Crosses for cytology were routinely made in Petri plates on synthetic crossing medium supplemented with 1%sucrose and2%agar(Davis,2000).For each cross, the protoperithecial parent wasfirst grown on medium for5days at25°C and then fertilized by adding conidia from the second parent.The H1-GFP and b-tubulin-GFP strains were examined both in homozygous and het-erozygous crosses,often made reciprocally.The develop-ing perithecia(unfixed,unstained)were dissected at12–24h intervals from3to10days post-fertilization on a glass slide in a drop of10%glycerol,lightly squashed un-der a cover glass and sealed with melted dental wax.
Asci were examined with an upright Nikon Micro-phot FX microscope equipped with epifluorescence and dark-field illumination.Afilter set specifically suited for sGFP imaging was obtained from Chroma (#41012:480/40nm excitationfilter,505nm dichroic mir-ror,510nm long pass emissionfilter).This combination offilters produced nofluorescence signal from any source other than from GFP.Low intensity back-light-ing through a dark-field condenser provided the neces-sary contrast and made ascus and ascospore outlines visible.Three objective lenses were used for observation and microphotography:Zeiss Neofluor10·(N.A.0.30), Nikon Plan20·(N.A.0.50),and Nikon Fluor40·Oil (N.A.1.30).For microphotography,a Nikon Coolpix 5000or5400digital camera was attached to the vid
eo port with adapters from Microscope World( www.microscopeworld).The5megapixels sensor in these cameras is capable of recording high-quality fluorescence images of asci,but it was often necessary to frame and focus cells on the cameraÕs LCD screen in brightfieldfirst,and then switch thefilter setup to fluorescence for2–8s manual exposures,depending upon the numerical aperture of the objective lens.
3.Results
3.1.Construction of sGFP plasmids for expression of translational fusions
We generated a series of versatile plasmids to express tagged fusion proteins assa,based on a system available visiae(Longtine et al.,1998).The plasmid backbone was designed as a simple cassette sys-tem that allows targeting to assa his-3locus and to accept various promoters,coding regions to be ex-pressed,and variousfluorescent marker genes(Fig.1 and data not shown).Building on results obtained with the strong ToxA promoter from Pyrenophora tritici-repentis(Freitag et al.,2001),we attempted to express translational fusions of Neurospora genes to sgfp driven by different Neurospora promoters.
When under the control of the Neurospora ccg-1 promoter,primary His+transformants with either hH
1+-sgfp+or Bml+-sgfp+showed GFP expression after $2–5days on minimal medium plates when visualized under thefluorescence stereo microscope.The native hH1promoter and the inducible Neurospora qa-2and arg-2promoters,however,did not result in visible H1-GFP expression(data not shown).For successful con-structs,typically>75%of all His+transformants showed strong GFP expression on the initial plate.Pri-mary transformants were transferred to slants and grown overnight at32°C,after which GFP was easily detectable in the aerial hyphae and developing macroco-nidia under the dissecting microscope.GFP expression was stable in strains kept at room temperature for a month and for at least a year in strains kept at À20°C.We observed increased autofluorescence in hy-phae from month-old cultures,which can obscure low levels of GFPfluorescence.Nevertheless,growth on
900M.Freitag et al./Fungal Genetics and Biology41(2004)897–910
fresh medium fully restored expression of H1-GFP and b -tubulin-GFP of strains kept in slants at À20°C for more than one year.
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Primary transformants are usually heterokaryotic.Thus,transformants were either crossed to obtain homokaryotic progeny or purified by serial conidial iso-lation (Davis,2000).The hH1+-sgfp +compone
GRAPHICALABSTRACTnt of strain N2276,for example,was purified by serial isola-tions to generate a homokaryon (N2276-1).Microscopic observations of heterokaryotic mycelia and conidia re-vealed that all nuclei showed green fluorescence,indicat-ing import of mature H1-GFP protein into all nuclei.The Neurospora ccg-1promoter is strongly induced by glucose deprivation or stress (McNally and Free,1988).Accordingly,we observed significant induction of fusion proteins after growth in liquid starvation med-ium.Cultures were grown for 2days in liquid medium supplemented with Vogel Õs minimal salts and 2%su-crose,harvested by filtration and the mycelial pads split in half.The ‘‘uninduced’’pad was quick-frozen in liquid nitrogen and stored at À80°C,while the remaining tis-sue was resuspended in Vogel Õs minimal salts without added carbon source for six hours.GFP was detected in the uninduced tissue,albeit at much lower levels than under induced conditions (data not shown).On solid medium,however,no increase in fluorescence was de-tected when strains were transferred to water agar after growth on Vogel Õs minimal salts and 2%sucrose or on
malt extract medium.The relative fluorescence of cul-tures grown on all solid media tested was comparable to that observed from induced liquid cultures,which suggests that the ccg-1promoter is activated,and re-mains active,when aerial hyphae are formed.
3.2.Nuclear organization and dynamics in hyphae mon-itored with H1-GFP
Nuclei labeled with H1-GFP fluoresced brightly (Fig.2).As expected (e.g.,Davis,2000),nuclei were typically absent in the tips (first 20–25l m)of growing hyphae (Figs.2A–C ;see Supplementary Movie 1at and www.sciencedirect ).This anucleate apical hyphal region was occupied by a Spit-zenko ¨rper in the very tip (as shown by double-labeling with FM4-64,Figs.2A and B )and mitochondria (as shown by double-labeling with DASPMI,Fig.2C ).Nu-clei were more plentiful in apical hyphal compartments than in older subapical hyphal compartments (cf.Figs.2A and E ).Rather than being diffusely localized in the nuclear matrix,H1-GFP stained nuclei unevenly and was found in stable fluorescent foci (Fig.2D ),which suggests differential association with chromatin.The shapes of nuclei in transport varied from oval to pear-shaped (Figs.2B–E ).A bright fluorescent region was often observed at the leading end of a moving nucleus (see Supplementary Movie 2at
www.
Fig.1.Construction of GFP expression plasmids for use assa .(A)Expression of the GFP variant gene (sgfp )was driven by the Neurospora ccg-1(P ccg-1)promoter (McNally and Free,1988).Plasmid pMF272(GenBank Accession No.AY598428)is a versatile cassette vector with a multiple cloning site (MCS)for construction of translational fusions of Neurospora genes to sgfp .Because the ATG start codon of sgfp was included,expression of this construct results in nucleoplasmic expression (Folco et al.,2003).The P ccg-1-sgfp cassette is contained on a pBM60backbone to allow his-3targeting by gene replacement (see Section 2;Margolin et al.,1997).The Nde I and Dra I restriction endonuclease sites are useful for linearizing constructs before transformation.(B)Partial maps of pMF280(AY598429)and pMF309(AY598430),carrying the histone H1(hH1)and b -tubulin (Bml )gene,respectively.
M.Freitag et al./Fungal Genetics and Biology 41(2004)897–910901
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