European Journal of Cell Biology 91 (2012) 930–937
Contents lists available at SciVerse ScienceDirect
European Journal of Cell
Biology
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /e j c
b
Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?
Sara Geraldo a ,b ,Anthony Simon a ,b ,Nadia Elkhatib a ,b ,Daniel Louvard a ,b ,Luc Fetler a ,c ,Danijela M.Vignjevic a ,b ,?
a
Institut Curie,Paris 75005,France b
UMR144/CNRS,France c
UMR168/CNRS,France
a r t i c l e
i n f o
Article history:
Received 9March 2012
Received in revised form 24July 2012Accepted 25July 2012
Keywords:
Focal adhesions 3D matrix Vinculin Collagen
Intravital imaging
a b s t r a c t
During metastasis,cancer cells breach the basement membrane and migrate through the stroma mostly composed of a network of collagen I ?bers.Cell migration on 2D is initiated by protrusion of the cell membrane followed by formation of adhesions that link the actin cytoskeleton to the extracellular matrix (ECM).Cells then move forwards by exerting traction forces on the adhesions at its front and by disassem-bling adhesions at the rear.In 2D,only the ventral surface of a migrating cell is in contact with the ECM,where cell–matrix adhesions are assembled.In 3D matrices,even though the whole surface of a migrating cell is available for interacting with the ECM,it is unclear whether discrete adhesion structures actually https://www.doczj.com/doc/cd7119528.html,ing high-resolution confocal microscopy we imaged the endogenous adhesome proteins in three different cancer cell types embedded in non-pepsinized collagen type I,polymerized at a slow rate,to allow the formation of a network that resembles the organization of EMC observed in vivo.Vinculin aggregates were detected in the cellular protrusions,frequently colocalizing with collagen ?bers,imply-ing they correspond to adhesion structures in 3D.As the distance from the substrate bottom increases,adhesion aggregates become smaller and almost undetectable in some cell https://www.doczj.com/doc/cd7119528.html,ing intravital imaging we show here,for the ?rst time,the existence of adhesome proteins aggregates in vivo.These aggregates share similarities with the ones found in 3D collagen matrices.It still remains to be determined if adhe-sions assembled in 3D and in vivo share functional similarities to the well-described adhesions in 2D.This will provide a major step forward in understanding cell migration in more physiological environments.
? 2012 Elsevier GmbH. All rights reserved.
Introduction
In order to escape the primary tumor and reach the blood ves-sels,cancer cells encounter different ECMs during their course of invasive migration.First they breach the basement membrane,a thin and dense sheet-like structure composed of a network of col-lagen IV and laminin (Yurchenco,2011).Then they migrate through the stroma composed of ?brillar collagens,proteoglycans and var-ious glycoproteins (Boot-Handford and Tuckwell,2003;Egeblad et al.,2010;Naba et al.,2012).
Cell migration is a continuous process,but for simplicity,it is often described in four individual steps.First,actin polymerization drives protrusions at the cell leading edge;second,those newly extended protrusions become stabilized by adhering to the extra-cellular matrix (ECM);third,the cell moves forward by exerting
?Corresponding author at:Equipe de Morphogenese et Signalisation cellulaires,UMR 144CNRS/Institut Curie,Institut Curie,25rue d’Ulm,75248Paris cedex 05,France.Tel.:+330142346366;fax:+330142346377.
E-mail address:danijela.vignjevic@curie.fr (D.M.Vignjevic).
traction forces at the adhesion points;and fourth,adhesions at the cell rear are released enabling translocation of the cell body.
However,most normal or cancer cells migrating in two-dimensional (2D)and three-dimensional (3D)substrates differ in their morphology and modes of migration (Petrie et al.,2009;Meyer et al.,2012).Cells migrating on planar 2D substrates,either plain or coated with ECM proteins such as laminin,collagen I or ?bronectin,develop broad and ?at protrusions called lamellipo-dia and ?nger-like protrusions called ?lopodia (Pollard and Borisy,2003;Vignjevic and Montagnac,2008).The same cells growing in 3D matrices are more elongated and while cellular protrusions that resemble lamellipodia and ?lopodia are still observed,they are smaller in size and in fewer numbers (Wirtz et al.,2011).This change in morphology is partly due to differences in dimensional-ity of 2D and 3D substrates.While in 2D only the ventral surface of a cell migrating is in contact with the ECM,in 3D the whole surface of a migrating cell is contacting ECM proteins.Those differences in cell morphology and cell–matrix interfaces have probably a major role in determining the mechanism and rates of cells migrating in different environments.
Adhesion to the ECM is mostly achieved by cell surface recep-tors called integrins.Intracellularly,integrins are connected to the
0171-9335/$–see front matter ? 2012 Elsevier GmbH. All rights reserved.https://www.doczj.com/doc/cd7119528.html,/10.1016/j.ejcb.2012.07.005
S.Geraldo et al./European Journal of Cell Biology91 (2012) 930–937931
contractile actomyosin bundles via an entire collection of proteins named integrin“adhesome”that have mechanosensory and sig-naling functions(Geiger and Yamada,2011).Ef?cient cell migration depends on the coordination of changes in the actomyosin intracel-lular machinery and dynamics of adhesions with the extracellular environment(Parsons et al.,2010).
Cells migrating on2D substrates assemble several types of adhe-sions.They differ in size,protein composition,dynamics and their location in the cell(Geiger and Yamada,2011).However,there is controversy in the?eld whether discrete adhesion structures actu-ally exist in cells migrating in3D matrices and,more importantly, in vivo.
While some of the reports describe distinct adhesion structures containing“adhesome”proteins such as vinculin,paxillin and zyxin in cells migrating in different3D matrices,others show that adhe-some proteins do not form distinct aggregates but are fairly diffuse in the cytoplasm(Cukierman et al.,2001;Tamariz and Grinnell, 2002;Li et al.,2003;Petroll and Ma,2003;Wozniak et al.,2003; Martins and Kolega,2006;Provenzano et al.,2009;Fraley et al., 2010;Deakin and Turner,2011;Hakkinen et al.,2011;Kubow and Horwitz,2011).Those studies used different matrices(cell derived matrices or collagen I of different concentrations and methods of extraction),and cells that were either fully embedded in3D matrix or just seeded on top(Harunaga and Yamada,2011).Since a detailed analysis of this controversy has already been published by Harunaga and Yamada(2011),we will only refer here to pos-sible explanations for those discrepancies.First,going away from the rigid glass coverslips,the elasticity of3D matrices increases, which could affect the assembly of distinct adhesions(Fraley et al., 2011;Harunaga and Yamada,2011).Second,the organization of the3D matrices can in?uence the assembly of adhesion struc-tures.In3D collagen matrices mostly consisting of short and thin ?bers,cell adhesions,which are in the micrometer range,cannot physically assemble on thin collagen?bers that are nanometers in diameter.However,more extensive collagen bundling would provide suf?cient width to allow assembly of adhesions.Third, over-expression of?uorescently labeled adhesome proteins could obstruct the visualization of distinct aggregates.Indeed,distinct adhesions were detected when?uorescently labeled adhesome proteins are expressed at low levels(Kubow and Horwitz,2011). Fourth,over-expression of some adhesion proteins,such as pax-illin,can even have an effect on the cell migration strategy in which adhesions are no longer detected(Deakin and Turner,2011). Finally,differences in resolution and sensitivity of the microscopy systems used in different studies could also provide an explanation for these discrepancies.
To address this controversy,here we combine imaging of endogenous adhesome proteins in thick3D in vitro matrices, using high-resolution confocal microscopy,with imaging of?uo-rescently labeled adhesome proteins in xenografts of living animals by two-photon laser-scanning microscopy.Aggregates of endoge-nous proteins were detectable in three different cancer cell types fully embedded in matrices made of collagen type I?bers.Intravital imaging of xenografted cancer cells expressing low levels of GFP-Paxillin or GFP-Vinculin allowed for the?rst time observation of aggregates of adhesome proteins in the living mouse.
Materials and methods
Antibodies
Human antibody against tubulin was from Recombinant Pro-tein and Antibody Platform(Institut Curie).Mouse antibody against vinculin was a kind gift from M.Gloukhova.Mouse anti-bodies against?-tubulin,paxillin and zyxin were from Sigma,Transduction Laboratories and Synaptic Systems,respectively.Cy2-conjugated secondary anti-human antibody was from Jackson. Alexa-conjugated secondary antibodies,Alexa-and rhodamine-conjugated phalloidin were from Invitrogen.
Cell culture
HCT116,CT26and MDA-MB-231cells(ATCC)were cultured in culture medium containing DMEM(Gibco)supplemented with10% (v/v)fetal bovine serum(FBS,Gibco).Cells were maintained at37?C in10%CO2humidi?ed air during culture.
Collagen I labeling
Rat tail collagen I(non-pepsinized;BD Biosciences)dissolved in acetic acid0.2%(v/v)was dialyzed overnight against labeling buffer(0.25M NaHCO3,0.4M NaCl,pH9.5)at4?C.Tetramethyl-rhodamine(TAMRA,Invitrogen),resuspended in DMSO according to the manufacturer’s instructions,was diluted in labeling buffer, mixed with collagen solution and incubated overnight at4?C with agitation.Free dye was removed by dialyzing the labeled collagen against labeling buffer overnight at4?C.TAMRA-labeled collagen was?nally dialyzed overnight against acetic acid0.2%(v/v)at4?C.
2D collagen I substratum
Glass coverslips were coated with rat tail collagen type I TAMRA-labeled mixed with unlabeled collagen(BD Biosciences)in a1:6 ratio diluted in0.02N acetic acid to achieve33?g/cm2.After1h at room temperature,coverslips were washed3times with PBS and HCT116and CT26cells plated at low density.Cells were maintained at37?C in10%CO2humidi?ed air for1or2days.
3D collagen I matrix
HCT116,CT26and MDA-MB-231cells embedded in3D collagen I matrices were prepared by mixing TAMRA-labeled collagen with unlabeled rat tail collagen type I in acetic acid in a1:6ratio with10×PBS and cells resuspended in DMEM,to achieve a?nal cell density of105cells/ml in2mg/ml collagen matrix.1N of NaOH was added to increase pH to7.4.All reagents were kept at4?C.100?l drops of collagen mix containing104cells were added to glass bottom dishes(World Precision Instruments)and collagen was allowed to polymerize by increasing the temperature to either room tempera-ture or37?C.After polymerization,suf?cient culture medium was added to cover the collagen/cells drops and maintained at37?C in 10%CO2humidi?ed air for1or2days.
2D and3D immuno?uorescence staining
2D–Cells were pre-extracted with extraction buffer(1%Tri-ton X-100,4%PEG40000in PEM buffer(100mM Pipes,pH 6.9,1mM MgCl2,1mM EGTA))supplemented with2?M Phal-loidin(Invitrogen)and2?M Taxol(Invitrogen)for30s at room temperature.After3quick washes with PEM buffer supple-mented with2?M Phalloidin and2?M Taxol,cells were?xed in4%PFA for20min at room temperature.After3washes with PBS,cells were incubated with primary antibodies against tubulin,paxillin,vinculin or zyxin for1h at room temperature. Cells were washed3times with PBS followed by incubation with the appropriate Cy2-or Alexa-conjugated secondary anti-body and Alexa-or rhodamine-conjugated phalloidin for1h at room temperature,and further washed3times with PBS before mounting.
3D–Cells were immunostained as described(Hakkinen et al., 2011)with modi?cations.Cells were simultaneously?xed and
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Fig.1.The cytoskeleton of CT26colon cancer cells in 2D and 3D.Immuno?uorescence spinning disk images of CT26cells labeled with phalloidin for F-actin (green)and tubulin antibody (magenta)plated on TAMRA-labeled collagen I (blue)coated-coverslips (2D)or embedded on TAMRA-labeled collagen I matrix (3D).3D image corresponds to x –y maximal projection of Z stacks of 62?m.Bottom row corresponds to zoomed views of the boxed regions in the middle 3D row.Some Z planes have been removed for clear visualization of single microtubules.Scale bar,10?m.
extracted with detergent extraction ?xative buffer (4%PFA,0.3%Triton X-100,5%sucrose in PBS)for 5min.For cytoskeleton stain-ing,buffer was supplemented with 2?M Phalloidin and 2?M Taxol.Cells were further ?xed with 4%PFA,5%sucrose in PBS for 30min
and incubated with primary antibodies against tubulin,paxillin,vinculin or zyxin for 45min at room temperature.After 3washes with 0.05%Tween 20in PBS,cells were incubated with the appro-priate Cy2-or Alexa-conjugated secondary antibody and Alexa-
or
Fig.2.Vinculin rich cell–matrix adhesions in MDA-MB-231breast cancer cells.Immuno?uorescence spinning disk images of MDA-MB-231cells labeled with vinculin antibodies (green)embedded on TAMRA-labeled collagen I matrix (magenta).Images correspond to x –y maximal projections of Z stacks of 6.6?m,8.2?m and 18?m,starting 0?m,40?m and 100?m away from the substrate bottom (as de?ned in the Materials and methods section),respectively.Far right columns correspond to zoomed views of the boxed regions.Scale bar,10?m.
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Fig.3.Vinculin rich cell–matrix adhesions in HCT116colon cancer cells.Immuno?uorescence spinning disk images of HCT116cells labeled with vinculin antibodies(green) and phalloidin for F-actin(blue)plated on TAMRA-labeled collagen I(magenta)coated-coverslips(2D)or embedded on TAMRA-labeled collagen I matrix(3D).3D images correspond to x–y maximal projections of Z stacks of8.8?m,15?m and16?m,starting0?m,40?m and100?m away from the substrate bottom,respectively.Far right columns correspond to zoomed views of the boxed regions.Scale bar,10?m.
rhodamine-conjugated phalloidin for30min at room temperature, and further washed3times with0.05%Tween20in PBS before mounting.
Microscopy and imaging processing
2D and3D cultures were imaged with an Inverted Confocal Spinning Disk(Roper Scienti?c/Nikon)equipped with60×/1.4NA (working distance130?m)and100×/1.4NA oil-immersion objec-tives and controlled by Metamorph imaging software(Universal Imaging).491nm,561nm and633nm lasers were typically used at20–30%power.Cells were imaged only in the center of colla-gen drops to avoid edge effects.Z slices were acquired at0.2?m intervals.The?rst Z slice of TAMRA-labeled collagen detection determined the distance from the substrate bottom.Z stacks were maximally projected using Metamorph Of?ine(Universal Imag-ing)to create2D images and further processed using Photoshop CS5(Adobe)using only standard contrast and intensity level adjustments.Intravital two-photon microscopy
Immunode?cient(Swiss/Nude)female mice of6–8weeks of age were obtained from Charles Rivers,France,maintained in a speci?c pathogen-free environment and all the experiments were carried out with the approval of the local authorities.CT26cells were transfected with pmTomato and GFP-tagged Paxillin or Vinculin(a kind gift from C.Ballestrem)by nucleofection(Lonza)according to the manufacturer’s instructions and kept in culture for20h after transfection.50?l of PBS containing approximately2×106pel-leted tumor cells were injected into the dermis adjacent to the deep dermal vascular plexus of6–8-week-old female SWISS/Nude mice using a30-G needle.After5days,subcutaneous tumors of typi-cally5–7mm diameter were formed.Mice were anesthetized using 100?l of anesthesia/analgesia solution per10g of mouse.Anes-thesia/analgesia solution consists of7.5mg/kg xylazine,40mg/kg ketamine and0.5mg/kg?unitrazepan in PBS.An incision of approx-imately2cm×2cm was cut around the tumor creating a dorsal skin?ap and exposing the tumor.Mice were maintained in a temperature-controlled environment during the experiment and
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Fig. 4.Vinculin rich cell–matrix adhesions in CT26colon cancer cells.Immuno?uorescence spinning disk images of CT26cells labeled with vinculin antibodies (green)and phalloidin for F-actin (blue)plated on TAMRA-labeled collagen I (magenta)coated-coverslips (2D)or embedded on TAMRA-labeled collagen I matrix (3D).3D images correspond to x –y maximal projections of Z stacks of 13.8?m,19.8?m and 22.4?m,starting 0?m,40?m and 100?m away from the substrate bottom,respectively.Far right columns correspond to zoomed views of the boxed regions.Scale bar,10?m.
sacri?ced at the end of the imaging session.Tumors were imaged by two-photon laser-scanning microscopy (TPLSM)using a LSM510Meta (Zeiss,Jena,Germany)coupled to a MaiTai DeepSee fem-tosecond laser (690–1020nm)(Spectra-Physics,Mountain View,CA,USA),using a Zeiss Achroplan 40×/NA1.0water immersion objective.Fluorescence channels were recorded simultaneously with a 555nm dichroic mirror in combination with 472/30(SHG),520/35(GFP),and 585/40(Tomato)bandpass ?lters.The excitation wavelength was 920nm.3D stacks were obtained for up to 250?m penetration depth at a step size of 2?m.Z stacks were processed using Imaris (Bitplane).Standard contrast and intensity levels were further adjusted using Photoshop CS5(Adobe).
Results and discussion
Although cancer cells need to penetrate the basement mem-brane to invade and migrate in the surrounding tissue,they never migrate in it,since it is a structure less than 100nm thick (Yurchenco,2011).Therefore,we decided to use collagen I for our studies as a more appropriate ECM substrate for cancer cell migration.
The organization of 3D collagen matrices strongly depends on the puri?cation method of collagen I,as well as on the density of col-lagen ?bers and experimental conditions used for polymerization (Sabeh et al.,2009;Wolf et al.,2009;Wolf and Friedl,2011).For example,if collagen is pepsinized during extraction,it will poly-merize in long bundles with big pore size between the bundles.On the contrary,non-pepsinized collagen extracted from rat tail polymerizes in more dense gels.Similarly,low density collagen results in the formation of large pore size networks while high density gives small pore size networks.Finally,slower polymer-ization rates,achieved either by an increase of temperature and/or pH or by increasing the matrix volume,increases bundle formation and pore size of the network (Raub et al.,2007).We ?rst exam-ined the organization of extracellular matrix in the living animal.Collagen bundles visualized based in their intrinsic property by sec-ond harmonic generation,revealed that the diameter of bundles vary from 0.44to 3.72?m (on average =1.15?m,n =20)(Suppl.Fig.1).To achieve a collagen network similar to one observed in vivo,we used different matrix volumes polymerized either at 37?C or at room temperature (25?C)of 2mg/ml ?uorescently (TAMRA)-labeled non-pepsinized rat tail collagen I (Suppl.Fig.1).
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Fig.5.In vivo imaging of paxillin and vinculin rich cell–matrix adhesions in CT26colon cancer cells in the mouse dermis.Intravital two photon microscopy of CT26cells co-expressing cytosolic Tomato (blue)and GFP-paxillin (top panels)or GFP-vinculin (bottom panels;green),and endogenous ECM (magenta)imaged by second harmonic generation,5days after injection in the mouse dermis.For paxillin,images correspond to x –y projections of Z stacks of 30?m and 22?m.For vinculin,images correspond to x –y projections of Z stacks of 48?m and 32?m.Far right columns correspond to zoomed views of the boxed regions.Scale bar,20?m.
Polymerization of 10?l drops at 37?C resulted in the formation of a dense collagen network with visible collagen bundles from 0.20to 0.72?m thick (on average =0.47,n =20).Increasing the volume to 100?l also increased the bundle thickness to 1.30?m (on aver-age =0.55,n =20).The closest results to the in vivo collagen were achieved by polymerizing 100?l drops of collagen at room tem-perature.These experimental conditions resulted in the formation of a collagen network consisting of a mixture of a few thin bundles and many thick bundles varying from 0.72up to 1.56?m thick (on average =1.12,n =20).The diameter of those bundles should allow assembly of distinct adhesion points.
The major limitation in detecting cell–matrix adhesions in living cells is the background ?uorescence introduced by over-expression of ?uorescently tagged proteins.The excess of ?uorescent proteins diffusely distributed in the cytoplasm can mask the aggregates of adhesome proteins.Indeed,only when ?uorescently labeled adhesome proteins are expressed at low levels distinct adhesions become clear (Kubow and Horwitz,2011).To avoid these and
other caveats introduced by protein over-expression,we used 3D immuno?uorescence assays to visualize endogenously expressed proteins.First,we validated ?xation and staining procedures using antibodies against proteins with characteristic localization in the cells,such as tubulin.Although cells show different morphology when plated in 2D vs.3D substrates,a distinct and well organized network of the microtubule cytoskeleton can be observed in both preparations in all tested adenocarcinoma cell lines:mouse colon a CT26(Fig.1),human colon HCT116and human breast MDA-MB-231(Suppl.Fig.2).
Next,using the same immuno?uorescence methods we stained for well-characterized endogenous adhesome proteins,such as vin-culin,paxillin and zyxin.It has been suggested that binding and close proximity of collagen ?bers to very hard surfaces,such as glass,can have a strong effect on the ?ber’s tension (Fraley et al.,2011).This increase in tension can be transmitted to the entire network,decreasing with the distance from the glass.Since cell adhesions are mechano-sensitive structures,changes in tension
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of the matrix can have an impact on their size,number,shape and composition(Parsons et al.,2010).To investigate if the cell distance from the glass coverslip affects visualization of adhe-sions,we imaged cells at discrete distances from the substrate bottom.At0?m,cells are contacting the glass coverslip with their ventral surface,while their dorsal surface is covered with colla-gen.At40?m and above100?m,although cells are completely immersed in the collagen matrix in both cases,tension exerted by the glass on the matrix differs and might,therefore,have an impact on cell adhesions.In MDA-MB-231cells,a highly invasive cancer cell line,all cells show distinct adhesion structures con-taining vinculin(Fig.2)and zyxin(Suppl.Fig.3),independently of the distance from the coverslip.Those aggregates were localized in cellular protrusions,frequently colocalizing with collagen?bers, strongly suggesting they correspond to adhesion structures in3D. Paxillin aggregates are also clearly present in cells up to40?m, but hardly observed in cells at100?m distance from the substrate bottom.
HCT116cells,a colon cancer cell line with slow turnover of focal adhesions(60min,our unpublished data),exhibit a very?at-tened morphology on2D and when contacting the glass coverslip in 3D preparations.Vinculin aggregates were clearly detected under these conditions,accumulating at the cell periphery both at the leading edge and at the rear of the cell(Fig.3).However,when HCT116cells are fully embedded in the collagen matrix,not only do they dramatically change their morphology,but also vinculin staining becomes diffuse.When in3D,HCT116cells show a ten-dency to acquire a round morphology and to form clusters of3–10 cells,with only one or two cellular protrusions.Vinculin can be mainly found diffuse in the cytoplasm,occasionally accumulating in small clusters at the tips of the cellular protrusions.Both pax-illin and zyxin show a similar distribution to vinculin in HCT116 cells yet less distinct,both in close proximity and far from the glass coverslip in3D(Suppl.Fig.4).
Using a mouse colon cancer cell line,CT26cells,with fast turnover of focal adhesion(10min,our unpublished data)we could also detect aggregates of vinculin as in HCT116cells(Fig.4).Never-theless,the cellular localization of these aggregates in CT26cells in 2D was not in the lamella and cell periphery as in HCT116cells,but in the cell body,resembling the shape of?brillar adhesions.When fully embedded in the3D matrix,CT26cells acquired a typical mes-enchymal morphology,generally extending one single protrusion from the cell body,tipped with many?lopodia and lamellipodia. Although vinculin was mainly seen evenly distributed in the cyto-plasm,some distinct aggregates could also be found at the tips of cellular protrusions(Fig.4).
Finally,we aimed to image cellular adhesions in the most phys-iological environment,the living animal,using intravital imaging of cancer cells injected into the deep dermis of mice.CT26were transiently transfected with GFP-tagged adhesome proteins,pax-illin or vinculin,together with Tomato to allow visualization of the cell shape.After the tumor has grown,both the number of expressing cells and levels of expression of GFP-tagged adhe-some proteins and Tomato were very low,allowing detection of adhesome aggregates in isolated cells(Fig.5).GFP-paxillin and GFP-vinculin were mostly diffuse in the cytoplasm of CT26cells, differing from the Tomato distribution only by their absence in the nuclear region.However,it was occasionally possible to distinguish several small aggregates of both GFP-paxillin and GFP-vinculin in cellular protrusions and close to the nucleus.This localization is similar to the endogenous proteins found in3D collagen matrices by immunostaining.Although these GFP aggregates were not always detected colocalizing with collagen bundles(Fig.5,top panel),they were not enriched in Tomato,suggesting that it is speci?c for the adhesome proteins and may,therefore,constitute cellular adhesive structures to the ECM in vivo.Conclusion
Historically,cell migration has been studied in2D substrates. Cell biologists have de?ned the migration process as a sequence of four individual steps,characterized by rounds of cell protru-sions and attachments to the ECM.Nevertheless,it has become increasingly clear that the dimensionality of the system plays a critical role on cell morphology and modes of migration(Meyer et al.,2012;Petrie et al.,2012).In3D matrices,the cell–matrix interface is greatly modi?ed compared with2D,since the whole cell surface is available to contact with the ECM.Detecting cell–matrix adhesions in3D matrices has proven to be a dif?cult task and has raised some controversy.The nature and thick-ness of the matrix,as well as the distance from the rigid glass coverslips,have been the core of this debate.The expression levels of over-expressed adhesome proteins are also a factor to take into account.Although distinct adhesion structures were not always observed in3D matrices,the individual adhesome pro-teins were still found to regulate cell migration(Fraley et al., 2010).
In this short report,we used thick matrices of collagen type I, polymerized at a slow rate,to allow the formation of a network primarily consisting of collagen bundles resembling the organi-zation of the ECM observed in the living animal.We show that distinct cell–matrix adhesions can be visualized in3D matri-ces using immunostaining of endogenous adhesome proteins.We imaged cells at discrete distances from the rigid substrate bot-tom to evaluate the effect of the matrix elasticity.However, there are some differences that appear to be cell type speci?c. While adhesions were detected in all MDA-MB-231cells indepen-dently of the distance from the coverslip,in colon cancer cells, as the distance from the substrate bottom increases,adhesion aggregates become smaller and almost undetectable.This sug-gests that the distance from hard substrates is an important factor for the formation of cell–matrix adhesion contacts.Nevertheless, it is possible that factors other than collagen bundle thick-ness,distance from hard substrates,and protein over-expression, represent signi?cant aspects for the discrepancies reported in adhesion observations in3D.The differences in cell morphol-ogy and the presence of adhesion aggregates in2D and3D matrices,demonstrate that it is risky to establish direct parallels between2D studies to what is happening in the physiological3D environment.
While thick3D collagen I matrices represent,so far,the best simpli?ed in vitro system to study cell migration in physiologi-cal ECMs,it lacks the complex architecture,cellular and chemical diversity found in the living animal.The ultimate goal of the cell migration?eld is to visualize and characterize the mechanism of cell migration in living https://www.doczj.com/doc/cd7119528.html,ing intravital imaging we show here,for the?rst time,aggregates of adhesome proteins in vivo. These aggregates seem to share similarities with the ones found in3D collagen matrices.In the following years we should be able to grasp if adhesions assembled in3D and in vivo share similar-ities such as molecular composition,dynamics and functions to the well-described adhesions in2D.This would provide a major step forward in understanding cell migration in more physiological environments.
Acknowledgments
We gratefully acknowledge PICT IBiSA Imaging Facility and Recombinant Protein and Antibody Platform(Institut Curie).This work was supported by ANR-09-JCJC0023-01(DV)and EMBO post-doctoral fellowship(SG).
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Appendix A.Supplementary data
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（小学语文）句型转换练习题集锦（一） 一、反问句与陈述句 有时为了表达的需要，可以把陈述句变为反问句，也可以把反问句变为陈述 句，它们的意思相同，语气有所不同。 （一）反问句变陈述句 1、先删去反问词（怎能、怎么、难道、哪里等等），有的句子可适当再加上“很”“都”等，使句子表达的意思更准确。 2、看句子里的有没有否定词“不”，有的给删去，没有的给加上。 3、反问语气词删去“？”变“。”。 2、红四团取得了长征中的又一次决定性的胜利。 3、汽轮发电机厂的工人师傅把一台台进口钻机熟练地安装好了。 4、语文老师给大家讲了一个关于小红帽打败大灰狼的故事。 四、修改病句 病句类型：语序不当、搭配不当、成分残缺或赘余、结构混乱、表意不明、不合逻辑。 （一）成分残缺 1、春天来了，越来越来暖和了。 2、洒在沙滩上，沙滩上一片银白。 3、学了《爱迪生》这一课受到了教育。 4、雷锋全心全意为人民服务的事迹。 （二）成分赘余 1、联欢会上，他首先第一个走上台表演节目。 2、奶奶的菜园里种了茄子、西红柿、黄瓜和蔬菜。 3、我经常阅读、看科幻小说。 4、松鼠喜欢把窝搭在高大的大树上。 (三)表意不明 1、李红有时候经常和张明在一起打球。 2、妹妹找不到爸爸妈妈心里很着急。 3、有人主张接受，有人主张反对，他同意这种主张。 5、全班同学都参加了这次公益活动，只有张刚没有参加。 （四）搭配不当 1、今天联欢会上的音乐和舞蹈真好看。 2、我很喜欢参加自然博物馆。 3、战士们冒着倾盆大雨和泥泞的小路前进。 4、战斗正在热烈地进行着。 （五）语序不当 1、我们学校师生全体去参观科技馆。 2、王丽在演唱会上唱了优美的一支歌。 3、美术和音乐这两门功课对我很感兴趣。 4、李华被同学们一定评为三好学生。 5、学校把“学雷锋积极分子”的名单已经公布出来了 五、运用修改符号修改下列病句。 1.中国的人口是世界上最多的国家。 2、他很果断，从来不听别人的意。 3、今天的家庭作业是读二遍课文和四道数学题。 4、星期天，我们来到刚建成的、崭新的儿童公园。 5、这家电冰箱厂生产的冰箱质量一年比一年进步。 6、一面面鲜艳的红旗，在和暖的春风中迎风飘扬。 7、全国各地给灾区人民运去了大批的药品、食品、面包和衣服。 8、多读课外书，可以使我们增长知识和写作水平。 9、刘小刚第一次出席市里举行的作文竞赛，他心里既高兴又紧张。 10、因为别人的意见不一定全对，我们所以要虚心听取。 六、把下列句子分别改为“把”字句和“被”字句。 (1)这个故事里的主人公迷住了我。 (2)大风卷起的尘土遮住了我的视线。 (3)经过一千多次的试验,爱迪生终于找到了适合做灯丝的材料。 他要去补习。（双重否定句） 他要去看书。（双重否定句） 1.我们遵守纪律。 改为双重否定句：我们不得不遵守纪律 改为反问句：难道我们能不遵守纪律吗？ 他要去看电影。（双重否定句） 把直述句改为转述句 （1）爸爸对妈妈说：“你去把行李拿来。” （2）小红说：“桂林山水太美了！你们想去看吗？”（3）齐威王对田忌说：“怎么，难道你还不服气？”（1）爸爸让妈妈去把行李拿来。 （2）小红说桂林山水太美了，她问我们想不想去看。（3）齐威王问田忌他难道他还不服气。 转述句专项练习 1.妈妈对小明说：“放学后，我来接你。” 2. 老师对我说：“我教你怎么写。” 3. 雨来摇摇头说：“我在屋里什么也没看见。” 4. 李楠小声告诉我：“我家在少年宫附近。” 5. 小姑娘说：“我要去北京，我要去看看北京的名胜古迹。” 6. 老师对王芳说：“学校让你明天出席区小学生座谈会。”
小学英语句型专项练习题改为否定句 1.He can fly a kite. 2.She can do this. 3.I can swim. 4.She can skate，too. 5.I can read. And I can write too. 6.You’re Mike. 7.They are cute and fat. 8.They are in the bedroom. 9.Jane is very hungry. 10.I’m hungry. 11.It’s fifty-five yuan. 12.They’re very tall. 13.These are some pigs. 14.I like cats， too. 15.We like toy cars. 16.I have some grapes. 17.We have thirty yuan. 18.Mikes has some toy monkeys.
19.She has some toy dogs. 20.Jack likes to eat a pie on the sofa， too. 21.He likes monkeys. 22.I’d like some tables. 23.We would like some cakes. 24.Mike would like some sweets， too. 25.Open the door. 26.Close the window. 27.Listen to me. 28.Those are bananas. 29.We have some dolls， too. 30.I have some cakes too.
高中英语书面表达 基本句型训练 【句型解析】所谓简单句就是由一个主语(包括并列主语) 和一个谓语(包括并列谓语)组成的句子, 即一个主谓关系的句子。 , 或者该用be时却没用。因此, 我们须注意：(1)英语句子通常要有谓语动词, 否则就不完整；(2)当句中已有谓语动词时, 若不是进行时态或被动语态, 一定不要再用be。 【句型试练】用基本句型翻译下列各组句子, 然后合并成一篇通顺自然的5句话的短文。 ●话题：心中有梦想 ·单句翻译： 1.David 7岁了。(主系表) 2.他有一个梦想。(主谓宾) 3.他想在天上飞。(主谓宾) 4.他做了一架纸飞机。(主谓双宾) 5.他完成了作业。(主谓宾) 6.他玩纸飞机。(主谓) 7.天上有一架飞机在飞。(there be句型) 8. 他的父亲鼓励他努力学习成为飞行员。(主谓宾宾补) 9. 他非常努力地学习。(主谓) 10. 十八年后他实现了他的梦想。(主谓宾) ·合并成文：【拓展练习】用基本句型翻译下列各组句子, 然后合并成一篇通顺自然的5句话的短文。 ●话题：爬山日记 ·单句翻译： 1. 爬山是一种很好的运动。(主系表) 2. 爬山有益于健康。(主系表) 3. 今天我和同学去爬西山。(主谓) 4. 我们是乘出租去的。(主谓) 5. 我们大约在早上八点到达那里。(主谓) 6. 有许多游客在爬山。(There be) 7. 我们很兴奋。(主系表) 8. 我们一刻没耽搁就开始爬山。(主谓宾) 9. 班长小明帮了我很大忙。(主谓宾宾) 10. 我是唯一的女孩。(主系表) 11. 我们中午到达山顶。(主谓宾) 12. 我们很累但很高兴。(主系表) 13. 男孩子们让我跳舞。(主谓宾宾补) 14. 我的表演让他们忘记了疲劳。(主谓宾宾补) ·合并成文：
小学语文句型练习题集锦（一） 一、反问句与陈述句 有时为了表达的需要，可以把陈述句变为反问句，也可以把反问句变为陈述 句，它们的意思相同，语气有所不同。 （一）反问句变陈述句 1、先删去反问词（怎能、怎么、难道、哪里等等），有的句子可适当再加上“很”“都”等，使句子表达的意思更准确。 2、看句子里的有没有否定词“不”，有的给删去，没有的给加上。 3、反问语气词删去“？”变“。”。 练习： 1、我们怎能忘记老师的淳淳教导？ ____________________________________ 2、那浪花所奏的不正是一首欢乐的歌吗？ ______________________________ ____________________________________ 3、这里的景色这么美，怎能不使我们流连往返呢？ ___________________________________________________________ 4、这点小事，难道还要妈妈担心吗？ ____________________________________ 5、大千世界，哪里没有野花的倩影呢？ ________________________________ __________________________ （二）陈述句变反问句 第一步与第三步正好相反，第二步一样。 练习： 1、我们不能因为学习任务重而不参加体育活动。 ____________________________________ 2、这幅画是我们班彩颖画的。 __________________________________________ ________________ 3、父母含辛茹苦地把我们养大，我们不应该伤他们的心。 ___________________________________________________________ 4、对少数同学不守纪律的现象，我们不能不闻不问。 ___________________________________________________________ 二、对话与转述的互换 练习： 1、外公对小英说：“我告诉你，这是中国最有名的花。” _____________________________________________________________________ 2、张强笑着对妈妈说：“李明今天生病了，我去帮他辅导功课。” _____________________________________________________________________ 3、李老师兴奋地说：“我们班战胜了四（1）班，获得了冠军。” _____________________________________________________________________ 4、王老师对小华说：“我把你的书包缝好了。” _____________________________________________________________________ 三、缩句 1、要知道写的是什么。 2、表示时间、地点、数量、方向、环境、修饰词语（的、地、得前面的词语）
2017中考英语句子翻译专项训练 他们正在讨论如何处理这个问题。 他习惯和朋友通过微信来交流。 为什么不上网搜寻一下信息呢? 这次，你最好不要拒绝参加这次会议。 . 你介意和我一起编首曲子吗？ 我忍不住吃光了所有的水果沙拉。 . 在工作中我们应该尽力避免犯同样的错误。 对于我们而言，学会和他人紧密合作是必要的。 自从她去了国外后，你们彼此还保持联系吗？ 请警告他不要在离开时任由水龙头一直开着。 你知道历史上第一只风筝是由木头做成的吗？ 依靠你自己是多么有意义啊！ 我们应该帮助残疾人而不是仅仅同情他们。 众所周知选择公共交通工具将有助于减少空气污染。 人类已经意识到保护野生动物的重要性了吗？ 他在向我解释病毒已经使一切都出错了。 为了写下你所见的东西，请随身带好笔记本。 这个科学家正忙于在公共场合做演讲。 我想知道你是否可以用英语描述一下你的日常生活。 环境污染如此严重，以致导致了各种疾病。 他建议我们接受新挑战并三思而行。 我的一位外国朋友来中国很多年了，所以他中文说得很好。去年你父亲多久去北京出差一次？ 我父母对我如此严格，以致我真的感到筋疲力尽。 我过去常常梦想成为一名世界闻名的音乐家。 他已经决定放弃工作去周游世界。 . 这位学生分发所有的杂志有困难。 我认为这双鞋子和我的不是同一个尺码。 当我们到达山顶时，我再也走不动了。 这个老人没有可倾诉的朋友，所以感到有点孤独。 . 据说这本书将被译成英语。 . 这本书给我许多关于如何改善我们生活的好建议。 我们相信谭盾的音乐会将给我们开启一个全新的世界。
我们认为打断别人说话是不礼貌的。 今天，这位着名的人士将根据他自己的经历来谈谈餐桌礼仪。教练们将使用什么方法来帮助他们实现梦想? 医生们尽快地给这个生病的男孩动了手术。 . 他把他的一生都奉献给了基础教育的发展。 这些大学生们已经下定决心为贫困地区的孩子们创办新学校了。应该采取更多的措施来关注每个细节。 如果你遵循这些小步骤，它们会对地球产生巨大的影响。 这套别墅太贵，我们买不起。 莫言在50多岁时被授予诺贝尔文学奖。 他的父母别无办法，只能承诺帮助他。 我怀疑他是否撒谎，并且是否值得和他谈一谈。 现在，由于空气污染，许多人宁愿住在乡下。 每当我们难以做出决策时，总盼望着您宝贵的意见。 . 我父母不允许我熬夜。 . 他不断练习并且在英语上取得了进步。 这位发明家从不灰心，最终成功地发明了这个最新的机器人。这位老师经常鼓励孩子们改善他们的发音。 . 这个女孩不仅对音乐感兴趣，而且有舞蹈天赋。 . 这位艺术家直到去世后才因他的艺术品而闻名。 去年他很自豪地为有线电视新闻网现场报道了这场比赛。 除非你工作达到高标准，否则你会落后的。 他看上去很不高兴。没有人知道这个访谈节目让他想起了谁。 . 令我惊讶地是，所有人都坚持认为这位作家和谋杀有关。 张艺谋是一位如此天生的导演，以致他在电影界起着重要作用。成龙已经花了很多年的时间参加许多的慈善活动， 风雨雷电会阻止飞机准时起飞。 . 女孩子从来不会嫌衣服多，因为她们希望吸引他人的关注。 在古镇的中心有上百棵老树。 这位女演员为了获得这个奖每天都花五个半小时练习舞蹈吗？众所周知现在老人的数量正变得越来越多。 他碰巧发现了一些有持续价值的东西。 . 外国人对传统的中国艺术很着迷，例如中国戏曲。 我起得很早为了能观看升旗仪式。 你认为谁是月球上行走的第一人?
小学英语语法：助动词do 的用法 1) 构成一般疑问句，例如： Do you want to pass the CET? 你想通过大学英语测试吗? Did you study German? 你们学过德语吗? 2) do + not 构成否定句，例如： I do not want to be criticized. 我不想挨批评。 He doesn't like to study. 他不想学习。 In the past, many students did not know the importance of English. 过去，好多学生不知道英语的重要性。 3) 构成否定祈使句，例如： Don't go there. 不要去那里。 Don't be so absent-minded. 不要这么心不在焉。 说明：构成否定祈使句只用do，不用did和does。 4) 放在动词原形前，增强该动词的语气，例如： Do come to my birthday party. 一定来参加我的生日宴会。 I did go there. 我确实去那儿了。 I do miss you. 我确实想你。 5) 用于倒装句，例如： Never did I hear of such a thing. 我从未听说过这样的事情。
Only when we begin our college life do we realize the importance of English. 只有在开始大学生活时我们才理解到英语的重要性。 说明：引导此类倒装句的副词有never, seldom, rarely, little, only, so, well等。 6) 用作代动词，例如： ---- Do you like Beijing? --你喜欢北京吗? ---- Yes, I do. --是的，喜欢。(do用作代动词，代替like Beijing.) He knows how to drive a car, doesn't he? 他知道如何开车，对吧? 助动词do 的用法是我们精心为大家准备的，希望大家能够多加练习，为以后学好英语打下坚实的基础!
英语基本句型写作练习一 分析下列句子成分并写出句型。 1. You should study hard. 2. She went home very late yesterday evening. 3. That morning we talked a great deal. 4. I wrote a letter last night. 5. I want to talk with you this afternoon. 6. He has read this book many times. 7. My brothers are all college students. 8. Mrs Brown looks very healthy. 9. Mr Johnson taught us German last year. 10. In winter, the days are short and the nights are long. 11. Grandma told me an interesting story last night. 12. Mary handed the wallet to the schoolmaster. 13. Would you please pass me the dictionary? 14. We call her Alice. 15. His parents named him John. 16. All of us considered him honest. 17．They pushed the door open. 18. There isn’t going to be a meeting tonight. 19. There was only a well in the village. 20. There is (are) a teacher of music and a teacher of art in the school.指出下列句子加粗部分是什么句子成分： 1. The students got on the school bus. 2. He handed me the newspaper. 3. I shall answer your question after class. 4. What a beautiful Chinese painting! 5. They went hunting together early in the morning. 6. His job is to train swimmers. 7. He took many photos of the palaces in Beijing. 8. There is going to be an American film tonight. 9. He is to leave for Shanghai tomorrow. 10. His wish is to become a scientist. 11. The meeting will last two hours. 12. They have carried out the plan successfully. 13. At the age of fifteen he became a famous pianist. 14. He showed the ticket to the conductor. 15．They have set the thief free.
英语句式结构专项练习 翻译练习： 1. 你应当努力学习。 2. 她昨天回家很晚。 3. 那天早上我们谈了很多。 4. 会议将持续两个小时。 5. 在过去的十年里，我的家乡已经发生了巨大的变化。 6. 这种事情全世界各地每天都在发生。 7. 1919年，在北京爆发了“五四”运动。 8. 每天八点开始上课。 9. 这个盒子重五公斤。 10. 五年前我住在北京。 11. 爱丽丝游泳游得很好。 12. 约翰的父亲昨晚去世了。 13. 秋天有些鸟飞到南方去。 14. 我的爷爷早晨起得很早。 15. 每天下午有许多学生到图书馆来借书。 1. You should study hard. 2. She went home very late yesterday evening. 3. That morning we talked a great deal. 4. The meeting will last two hours. 5. Great changes have taken place in my home town in the past ten years. 6. Things of that sort are happening all over the world every day. 7. The May Fourth Movement broke out in Beijing in 1919. 8. Classes begin at eight every day. 9. This box weighs five kilos. 10. I lived in Beijing five years ago. 11. Alice swims very well. 12. John’s father died last night. 13. In autumn, some birds fly to the south. 14. My grandfather gets up early in the morning. 15. Every afternoon a lot of students come to the library to borrow books. 翻译练习： 我不信任那个人。（believe in) 他指出了我的作文中的错误。 (point out) 圣诞节我们将去看望外籍教师。(call on ) 你们必须在课后把练习本交上来。(hand in)
一、按要求写句子。 １、把“把”字句改成“被”字句。 疲劳和干渴，把它们折磨得有气无力。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ２、把“被”字句改成“把”字句。 我的错误马上被朋友们纠正了。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ３、改变词序，意思保持不变。 出金华城大约５公里到罗甸。＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 雨从早到晚哗哗哗哗地下个不停。＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ４、改变词序，改变句意。 那些来南京的客人都很热情。＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 这几道题你全没有做对。＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ５、把反问句改成陈述句。 我们哪里有近道，还不和你们是一条道？ ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 毒刑拷打算得了什么？死亡也无法叫我开口。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 都是你自己找的，我怎么帮得了你的忙？ ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ６、把陈述句改成反问句。 猫的性格实在有些古怪。＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 在这个长满了红锈的鱼钩上，闪烁着灿烂的金色的光芒。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ７、把直接引语改成间接引语。 周瑜长叹一声，说：“诸葛亮神机妙算，我真不如他。” ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 山姆握着珊迪的手说：“我会永远照顾你的。” ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 张林告诉我：“要是星期日下雨，我们就不去看电影。” ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ８、把间接引语改成直接引语。 妈妈严肃地对我说，马上就要期末考试了，我一定要认真复习，不要辜负老师和她对我的一片期望。 二、照样子写句子。 １、例：李老师推开门走进教室。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ２、例：夜空的繁星，仿佛碧波上撒满的宝石。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ３、例：井冈山是中国革命的摇篮。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ４、例：敌人使用了燃烧弹，邱少云的周围成了一片火海。 ＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿。 ５、例：天空的星星快活地眨着眼睛。
中考英语重点句型专项训练 1. 毕竟，这个孩子太小还不能上学。too...to... 2.只要我努力我的梦想总有一天会实现的。come true 3. 她过去靠卖书为生。live on 4. 我家离学校不远。far from 5.有一些人在河边钓鱼。 6. 我长大后想成为一名老师。grow up 7. 这把尺子是那把尺子两倍长。twice 8. 我乐于和平地生活。in peace 9. 我乐于和别人交朋友。make friends with 10. 从今以后，我不但要更加努力学习还要尽力取得更好的成绩。try my best 11. 我们一做完饭就应该把火灭掉。as soon as 12. 我以前在电脑游戏上花费了很多时间以至于我在学习上失去了兴趣。 so...that... 13. 明天我一到美国就给你打电话。as soon as
14. 我很高兴照顾我喜欢的人。care for 15. 在我回家的路上，我碰见了我的老朋友。on my way 16. 每天直到我回来，我父母才睡觉。not...until... 17. 听说你现在过得很愉快，我很高兴。enjoy oneself 18. 长城是世界八大奇迹之一。one of 19. 我经常看见他匆匆忙忙的去上学。in a hurry 20. 我的父母总是叫我别嘲笑别人。laugh at 21. 他既不喜欢游泳也不喜欢跑步。neither...nor... 22. 他学习不如我努力。as...as... 23. 当然，我们应该学会如何与别人和睦相处。get on well with 24. 我奶奶以前反反复复给我讲过这个故事。over and over again 25. 就我所知，北京因它悠久的历史和丰富的文化而闻名于世。be famous for 26. 我们应该阻止人们砍伐树木。stop ... from... 27. 我们应该尽力阻止动物灭绝。stop ... from...
be动词，情态动词，助动词do/does的用法区别及练习 助动词，顾名思义就是帮助动词完成疑问及否定的，本身没有什么含义。主要的助动词有be,do,will,have等，其用法详述如下： 一、⑴由连系动词am，is，are构成的句子：变一般疑问句时把am，is，are提到句子的前面，句尾用问号即可。变否定句时直接在am，is，are后面加not即可。例如： 肯定句:He is a student. 一般疑问句: Is he a student? 否定句: He is not a student. 画线提问: 对he提问: Who is a student? 对a student 提问: What is he? (2)was 是am,is的过去式，were是are的过去式，若句子中有以上两词时，变疑问句及否定句方法与（1）相同。 二、(1) 由情态动词can, may,will ，shall等构成的句子: 变一般疑问句时把can, may,will ，shall提到句子的前面,句尾用问号即可.变否定句时直接在can,may,后面加not即可. 例如: 肯定句: She can swim. 一般疑问句: Can she swim? 否定句: She can not swim. 画线提问: 对she提问: Who can swim? 对swim提问: What can she do? （2）could,might,would,should是can,may,will,shall的过去式，若句子中有以上两词时，变疑问句及否定句方法与（1）相同。 三、（1）由行为动词构成的句子: 需要加助词do或does. 变一般疑问句时把do/does放在句子前面. 变否定句时把don’t/doesn’t放在动词的前面。要注意观察动词的形式并对号入座。一般疑问句和否定句的动词三单式都要变回原型。 play-----do plays-----does 例如: 肯定句: They play football after school. He plays football after school. 一般疑问句: Do they play football after school? Does he play football after school? 否定句: They don't (do not) play football after school. He doesn’t’ play football after school. 画线提问: 对they/he提问: Who plays football after school? 对play football提问: What do they do after school? What does he do after school? 对after school提问: When do they play football? When does he play football? （2）did是do和did的过去式，变一般疑问句时把did放在句子前面. 变否定句时把didn’t 放在动词的前面, 要注意观察动词的形式并对号入座。一般疑问句和否定句的动词都要变回原型。
（小学语文）句型转换练习题集锦 一、反问句与陈述句 （一）反问句变陈述句 （1）将肯定句中的肯定词（是、能、会、有等）改为否定词（不是、不能、不会、没有等）。 （2）将否定句中的否定词（不是、不能、不会、没有等）改为肯定词（是、能、会、有等）。 （3）在反问句上删除反问语气词“怎么、难道”和句尾疑问助词“呢、吗”。 （4）句末的问号（？）改为句号（。） 【例1】我们怎么能言而无信呢？→我们不能言而无信。 【例2】欧阳海舍身拦惊马的英雄事迹，怎么不令我们感动呢？→欧阳海舍身拦惊马的英雄事迹， 令我们感动。 练习： （二）陈述句变反问句 （1）将肯定句中的肯定词（是、能、会、有等）改为否定词（不是、不能、不会、没有等）。 （2）将否定句中的否定词（不是、不能、不会、没有等）改为肯定词（是、能、会、有等）。 （3）在肯定词或否定词前面加上“怎么、难道”等反问语气词。 （4）句尾加上疑问助词“呢、吗”等，句末的句号（。）改为问号（？）。 【例1】：雷锋叔叔是我们学习的榜样。→难道雷锋叔叔不是我们学习的榜样吗？ 【例2】：我们不能浪费时间。→我们怎么能浪费时间呢？ 练习： 1、我们不能因为学习任务重而不参加体育活动。 2、没有革命先辈的流血牺牲，就没 有我们今天的幸福生活。 __________________________________________ 3、父母含辛茹苦地把我们养大，我们不应该伤他们的心。4、对少数同学不守纪律的现象， 我们不能不闻不问。 ___________________________________________ （三）把直述句改为转述句（直接引语改间接引语） 一、改变人称；二、去掉说后面的冒号和双引号，改为逗号；三、指示代词（这、那）和倾 向性动词（来、去）要根据语境发生变化。四、弄懂句子意思，知道谁对谁说话。五、提示语在 中间或在句末的句子，把提示语调整到句首可按以上方法进行改动。六、问句要改成陈述句。七、 看句子的内容，有些句子中个别的和少量的文字可做改动，但不能改变意思。 （1）A对B说话，然后B转述给第三个人听妈妈对我说：“我明天还要去开会。”改为：妈妈 对我说，她明天还要去开会。 （2）A对B说话，然后C转述给第四个人听。妈妈对爸爸说：“我明天要去开会。”改为：妈 妈对爸爸说，她明天要去开会。 （3）直述句提示语在前面。妈妈说：“我明天还要去开会。”改为：妈妈说，她明 天还要去开会。 （4）直述句提示语在中间或者在后面。 A、“工作太多了。”妈妈说，“我明天还要去开会。”改为：妈妈说，工作太多了，她明天还 要去开会。 B、“我明天还要去开会。”妈妈说。改为：妈妈说，她明天还要去开会。 （5）第二个人转述（转给第三个人）中的人称代词的变化。 A、妈妈对我说：“我明天要去开会。”改为：妈妈对我说，她明天要去开会。 B、妈妈对我说：“你要好好学习。”改为：妈妈对我说，让我好好学习。 （6）第三个人转述（转给第四个人）中人称代词的变化。 妈妈对爸爸说：“你去买点菜。”改为：妈妈对爸爸说，让爸爸去买点菜。 （7）指示代词的变化（这，那） A、叔叔在信里对我说：“有时间我一定到你那儿玩。”改为叔叔在信里对我说，有时间他 一定到我这儿玩。 B、他对我说：“这是我的铅笔。”改为：他对我说，那是他的铅笔。
英语句型转换专项练习 ①：一般疑问句 陈述句改一般疑问句方法： ①把be动词（am is are）和情态动词(can, may, must...) 提前，放到句首，其它照写。遇I/we→you, my→your. Some→any. 句号→问号（？） 例如：陈述句: They are in the park. He can play the guitar.. 一般疑问句: Are they in the park? Can he play the guitar? 练习：把下列句子变成一般疑问句 1. I am listening to music. 2. Mike is a student. 3. Mary can clean the classroom. 4. They are in the zoo. 5. There are some flowers in the vase. 6. This is my sister. 7. We are sweeping the floor. ②借助助动词do/does.第三人称单数用does,其余人称用do，放到句首，其他照写。特别记住：前面用does,后面的动词一定还原成原形。遇I/we→you, my→your. Some→any. 句号→问号（？） 例如：陈述句: I like the ducks. He likes the dogs. 一般疑问句：Do you like the ducks? Does he like the dogs? 练习：把下列句子改为一般疑问句。 1. We need some masks. 2. They like making the puppet. 3. Su Hai and Su Yang live in a new house. 4. I put a book on my head. 5. They sing “In the classroom” together. 6. We play basketball on Sundays. 7. Tom likes listening to music. 8. I have a telescope. 9. Mike has a pot. 10.She has some blankets.
写作基本句型训练（二） 学习内容：so…that/such….that/ in order to…/ in order that…/ too…to…/ …enough to do… 教学步骤： Step1.学习下列句子并翻译成中文 1. He was so excited that h e couldn’t say a word. 2. The little boy saved every coin so that he could buy his mother a present on Mother's day. 3. He is so clever a boy that everybody likes him. 4. He is such a clever boy that everybody likes him. 5. Their performances were humorous enough to be copied by other. 6. Others think the earth got too much hot for the dinosaurs to live on any more. 7. He ran fast in order to catch the train. 8.I get up early in order that i can catch the early bus. 归纳出以上句型的意思： s o…that:_____________________________ so that:________________________ s uch…that:__________________________ enough to do:____________________ t oo… to:____________________________ in order to/that:___________________ Step2.学习难点 1.so…+adj/adv…+that…:如此…以致 …so that…:引导目的状语从句（句中含有情态动词）引导结果状语从句（句中不含情态动词） They got so excited that they cried out.（结果：因此） The little boy saved every coin so that he could buy his mother a present on Mother's day.（目的：为了） 2. …so +adj/adv+a +名词+that…=…such+a+adj/adv+名词+that….:如此…以致My father was so hard-working that he always forgot his meals. =My father was such a hardworking man that he always forgot his meals. =My father was so hardworking a man that he always forgot his meals. 3…to+adj/adv…+to :太…以不能 He is too young to look after himself. He got up too late to catch the first bus. 注意：①此句型本身已含有否定意思，句中不需再另加否定词。 如：He is too young to can’t look after himself.(×) ②如果too后面的形容词为happy, ready, willing, eager, anxious等表心理状态的词时，后面的动词表肯定：I’m just too eager to know the result.我只是太想知道结果了。 4.当that 引导的结果状语从句为肯定句时，so…that…=be…enough to do转换。当that 引导的结果状语从句为否定句时，so…that…==too…to..==be not…eno ugh to do
小学语文强化训练：句式练习 班级__________ 姓名__________ 得分__________ 时间：40分钟满分：100分 一、下列句子属于什么句型？把相应的字母填在括号里。(8分) A．祈使句B．陈述句C．疑问句D．感叹句 1．这次，我看到了草原。那里的天比别处的更可爱。( ) 2．记者，你见过电灯吗？( ) 3．寡妇的日子真困难啊！( ) 4．要好好听老师的话啊！( ) 二、照样子写句子。(48分) 例1：大熊猫吃竹叶。 胖乎乎的大熊猫津津有味地吃鲜嫩的竹叶。 1．郝副营长望着夜空。 ________________________________________________________ 2．人们跑向木桥。 ________________________________________________________ 例2：宽阔的礼堂里坐满了前来观看文艺演出的观众。 礼堂里坐满了观众。 3．高山的崖缝里冷得像冰窖一样。 _______________________________________________________ 4．旧钢琴的前面坐着一个十六七岁的姑娘。 ________________________________________________________
例3：遇到困难，我们难道应该退缩吗？ 遇到困难，我们不应该退缩。 5．贝多芬难道不是一个伟大的音乐家吗？ ________________________________________________________ 6．我订的计划难道不是最全面、最合理的吗？ ________________________________________________________ 例4：小红军对陈赓说：“我还要等我的同伴呢。” 小红军对陈赓说，他还要等他的同伴呢。 7．小丽对哥哥说：“我不过随便说说罢了。” ________________________________________________________ 8．李林的哥哥对我说：“今天太冷了，我不想出去。” ________________________________________________________ 三、按要求写句子。(20分) 1．同学们下了决心，非要登上山顶不可。(改为肯定句) _________________________________________________________ 2．群众都把头上的帽子、手里的报纸和别的东西抛上天去。(改为“被” 字句) _________________________________________________________ 四、阅读短文，完成练习。(24分) 无边的回忆 席慕蓉 ①我有一双塑胶的拖鞋，是在出国前两年买的，出国后又穿了五
七年级英语句型转换专项练习 1. I often help my parents. (改为一般疑问句) ________ you often ________ your parents? 2. He usually goes to the park by bus.(对画线部分提问) ______ ______he usually______ to the park ? 3. They usually walk to school.(主语用she改写句子) ______ usually ______to school. 4. Mr.Zhou often drives a car to work.(改为同义句) Mr.Zhou often goes to school ______ ______. 5. Do you usually come here by bus?(改为同义句) Do you usually ______ a_______ here? 6. She often does her homework at home.(改为一般疑问句) ______ she often ______ her homework at home? 7. Kangkang often rides a bike to school.(同义句转换) Kangkang often_____ _____ _____ _____ ______ . 8. Maria sometimes goes to the supermarket by subway. (对画线部分提问) ______ _____ Maria sometimes go to the supermarket? 9. Yu Jing usually plays computer games after school. (同上) _____ _____ Yu Jing usually ______ after school ? 10. They go to the library twice a week. (同上) _____ _____ _____they go to the library? 11. He is looking for his soccer. (改为一般疑问句) _____ _____ looking for his soccer? 12. They are making cards now.(改为否定句) They ______ ______ cards now. 13. She is having an English class. (对画线部分提问) ______ ______ she ______ ? 14. The dining hall is at the back of the school.(对画线部分提问) ______ ______ the dining hall? 15. They are talking with their English teacher. (改为同义句) They are ______ ______ their English teacher. 16. I want to work in China because I love China and its people.(对画线部分提问) ______ ______you want to work in China? 17. It's Tuesday today. (对画线部分提问) ______ ______ is it today? 18. The class is over at eleven o'clock. (对画线部分提问) ______ ______ ______ the class over? 19. We are having an English class. (对画线部分提问) ______ ______ are you having? 20. They have seven lessons every day. (对画线部分提问) ______ ______ lessons do they have every day? 21. There is a picture on the wall. (改为否定句) ______ ______ a picture on the wall.