LAB 4: Biological Membranes

Transcription

1 BIO 26: ENERGY FLOW IN BIOLOGICAL SYSTEMS LAB 4: Biological Membraes I. INTRODUCTION Membraes are aother molecule that make life possible. Most importatly, they provide a compartmet for cells, separatig the cytoplasm (the material withi a cell), with its multitude of molecules ad structures, from the outside eviromet. Most itracellular processes, especially ezymecatalyzed reactios, deped o a stable iteral eviromet (i terms of both molecular cocetratios as well as ph). Beyod beig a wall, membraes both regulate ad are the site of a whole host of importat biochemical reactios. The cell's membrae helps regulate the itracellular eviromet by regulatig what types of molecules go i ad out of the cell. Furthermore, may life processes are mediated by membrae-boud proteis. I this lab we will examie several characteristics of biological membraes, ad physical forces actig upo a cell's membrae. The simplest self-assemblig aggregate is a micelle, a small droplet with the hydrophilic heads o the outside ad the hydrophobic tails o the iside. This most commoly forms with amphipathic molecules with a small hydrophobic regio, such as ioized fatty acids with a sigle hydrocarbo tail. Phospholipids, however, do ot ofte form micelles because the two hydrocarbo tails are too bulky to fit i the iterior of a micelle. Istead, phospholipids usually form a thermodyamically more stable lipid bilayer. The resultig aggregate is a liquidfilled balloo called a liposome, with the wall of the liposome composed of the lipid bilayer (see Figure 4.2). Both sides of the II. MEMBRANE THERMODYNAMICS I oe sese membraes are very simple they are a double layer of phospholipids. As you leared i lecture, phospholipids are amphipathic molecules composed of a polar phosphate head group ad two opolar hydrocarbo tails (see Figure 4. at right). Whe amphipathic molecules are added to water they ca self-assemble ito aggregates. This self-assembly occurs maily due to hydrophobic iteractios (what are these iteractios?). The polar water molecules repel the hydrophobic tails, with the tails tedig to become closely packed with oe aother. Figure 4.. Structure of phospholipids. (a). Structural formula ad (b) space-fillig model. Phospholipids ca vary i the idetity of the head group (i this case a cholie) ad differeces i the two hydrophobic tails, each havig a carbo backboe ad hydroges attached. The kik i oe of the tails is due to a double bod. Figure adapted from Campbell ad Reece (2002) "Biology", 2 d ed. lipid bilayer are bouded by water, with the 4-

2 Bio 26 Lab 4 Membraes o-polar tails i the ceter of the bilayer. The stability of the bilayer is the result of two factors: first, water molecules are released from iteractig with the o-polar tails, which are i the ceter of the bilayer; ad secod, va der Waals forces betwee the tails favor the closely-packed bilayer arragemet. The complexity of membraes is due to other molecules preset i the lipid bilayer, such as cholesterol, glycolipids (lipids with a sugar group attached to the hydrocarbo chai), ad proteis. I this lab we will be examiig the characteristics of the lipids i the bilayer ad the behavior of a trasmembrae protei chael called aquapori. Polar Head Nopolar Tails III. MEMBRANE PERMEABILITY Biological membraes are lipid bilayers that compose the boudaries of cells. These barriers prevet molecules geerated i the cell from leavig the cell ad keep uwated molecules out. Lipid compoets of the membrae determie the permeability of the membrae itself. I particular, membraes have very low permeability to ios ad large polar molecules. Water, though a polar molecule, ca move across membraes due to its small size, high cocetratio ad lack of a complete charge. I geeral, the more opolar a molecule, the more readily it crosses a membrae. Note also that there are a variety of membrae proteis that trasport molecules (such as ios) that would ot otherwise be able to cross the membrae, though we wo't be lookig at these i lab today. What we will do is use red blood cells to ivestigate the permeability of cell membraes. Red blood cells are a good model because they are easy to obtai, they are uiform i size ad it is relatively easy for us to determie whe they udergo Figure 4.2. A lipid bilayer. Polar Head hemolysis (cell rupture). You will first look at red blood cells uder the microscope to observe the chages i cell shape which take place i hypertoic ad hypotoic solutios. The you will perform a secod experimet comparig the rate of hemolysis with differet compouds i solutio. As metioed above, water ca move across membraes; however its movemet through a lipid bilayer is relatively slow. I may tissues, water moves across the membraes very quickly due to the presece of pore-formig proteis called aquaporis. (Aquaporis were discovered i 99 by Peter Agre, ad he wo the Nobel Prize i Chemistry for his research i 2003 see Presto et al. 992, which is available o the lab web page.). Each aquapori (AQP) is a 4-2

3 Bio 26 Lab 4 Membraes a. b. Figure 4-3. Structure of Aquapori- i huma red blood cells. Aquapori is composed of six - helices that spa the membrae. (a). A represetatio of the six-helix barrel viewed parallel to the bilayer with the lies idicatig the approximate axes for the six helices (labeled A-F). The arrows idetify the ceter of the lipid bilayer. (b). Cross-sectio of a aquapori molecule that shows the passage for water i the ceter. Images adapted from: (a) Cheg et al. 997 Nature 387: ad (b) Murata et al Nature 407: trasmembrae protei with a passage i the ceter that allows water to pass through (see Figure 4.3). Each molecule is a moomer with six α-helices that form a barrel-shaped passage through the membrae. Aquaporis are preset i the cell membraes of red blood cells, as well as other water permeable membraes foud i the epithelial liig of itesties ad kideys. Whe we observe the effect of ioic cocetratios o red blood cells, we are idirectly observig the activity of the trasmembrae aquaporis. IV. OSMOLARITY differece i solute cocetratio produces a cocetratio gradiet across the orgaism's membrae. This gradiet is a kid of order, i the sese that it is ot radom. Ad if you Orgaisms ad their compoet cells ofte fid themselves aqueous eviromets with differig solute cocetratios. For example, the protozoa that we saw i lab earlier commoly occur i freshwater eviromets with low solute cocetratios, much lower tha that foud withi the orgaisms. This 4-3 Figure 4-4. Solute particles ca diffuse across a semi-permeable membrae from a area of high cocetratio to a area of low cocetratio. This diffusio will occur util solute cocetratio is equal o both sides of the membrae.

4 Bio 26 Lab 4 Membraes recall the secod law of thermodyamics, all process processes occur so as to icrease radomess (etropy). So there will be pressure, i this case called osmotic pressure, that will ted to act i such a way to equalize the solute cocetratios o each side of the membrae. There are two ways to equalize the solute gradiet. First, the solutes ca move across the membrae this process is called diffusio ad is show i Figure 4.4. Secod, water ca move across the membrae i a process called osmosis this process is importat whe water ca move across the membrae, but the solutes caot. Note that i both diffusio ad osmosis, particles move i order the equalize the solute cocetratio across the membrae. Osmolarity is a measure of the amout of solute preset i solutio. This cocept does ot take ito accout differeces betwee molecules which ioize i solutio, such as NaCl, ad those which do ot, such as sucrose. A M solutio of NaCl (each molecule of which dissociates ito two particles, Na + ad Cl - ) has a cocetratio of 2 Osm (osmolar = osmoles/l). Note that a 30 mm solutio of CaCl 2 has a osmolarity of 90 mosm because each molecule of CaCl 2 dissociates ito three particles i solutio. For a substace like sucrose, which does ot ioize, the osmolarity of a solutio is the same as the molarity. There are three terms used to compare solute cocetratios across a membrae. Isotoic (or isoosmotic) refers to a solutio havig the same solute cocetratio as aother solutio. If a cell is placed i a isotoic solutio, there will be o et movemet of water across the membrae. I medicie, itraveous fluids must be give at a cocetratio that is isotoic with blood (ormally 296 ±5 mosm). A solutio with a lower osmolarity that aother solutio is said to be hypotoic (or hypoosmotic), whereas a solutio with a higher osmolarity is hypertoic (or hyperosmotic). Note that all of these terms are relative terms. V. MICROSCOPIC OBSERVATIONS OF RED BLOOD CELLS I isotoic solutios, red blood cells (RBC's) are bicocave disks with a remarkably uiform diameter (7μm for huma RBCs). This shape allows them to fold up slightly as they squeeze i sigle file through capillaries. The cell membrae has a relatively fixed surface area. As the cell volume decreases i a hypertoic solutio, the cell membrae wrikles, ad the RBC takes o a created appearace. Coversely, as the cell volume icreases i a hypotoic solutio, the RBC will first swell ad lose its bicocave shape, the as the membrae itegrity becomes compromised, the itracellular cotets spill out of the cell (called hemolysis). I this experimet you will observe microscopically the effect of solutios of various osmotic stregths o the gross appearace of RBCs (we are usig sheep blood). These qualitative observatios of RBC behavior should led further support to the hypothesis that biological membraes are semipermeable. Please wear purple itrile gloves whe workig with blood. 4-4

5 Experimetal Procedure: Bio 26 Lab 4 Membraes Repeat the followig procedure with 3 differet cocetratios of sucrose: First 300 mm (this is iosotoic with the RBC cytoplasm), the 600 mm (hypertoic), the 00 mm (hypotoic). If you have three people i your lab group, have each perso i your group ca do oe of the solutios, ad the look at each other's slides. Use the Niko Alphaphot microscopes for this exercise.. Place a small drop of 2% RBC suspesio o a microscope slide. 2. Add medium drop of the Sucrose solutio directly o top of the RBC suspesio. 3. Add a cover slip ad examie the slide immediately through the compoud microscope. Focus first at low power, the 0x, the 40x. If the RBCs are crowded too closely together to see idividual cells clearly, repeat the procedure with a smaller volume of RBC suspesio. You should be able to describe the appearace of the RBCs uder each coditio ad discuss what happeed i each case. Why do RBCs chage shape whe the differet solutios are added? What is happeig to water i these three cases? VI. RBC PERMEABILITY IN DIFFERENT SUBSTANCES As you observed i the exercise above, the red blood cell (RBC) is ormally a flatteed, bicocave disk. Whe the RBC's are placed i a hypotoic solutio, water moves ito the cells through aquapori chaels. The cells will iitially swell ad become spherical. As more water moves ito the cells, membraes will stretch ad hemoglobi will begi to leak out. Evetually, the cell membrae will burst, leavig behid the empty cells, or "ghosts". If you look at the 2% blood solutio from the previous exercise, you will otice that dilute blood is opaque (i.e., it is ot trasparet). However, whe the cells burst or "hemolyze", the blood mixture will become a trasparet red color. The membraes o loger block light ad the hemoglobi goes ito solutio, titig the liquid red. I this experimet, you will observe the time of hemolysis (i.e., how log it takes for red blood cells to lyse or burst) i differet solutios. These substaces (show below) iclude a series of salt (NaCl) solutios at differet cocetratios ad alcohols with differet lipid solubilities. A. Distilled water (DH 2 O) B M NaCl C M NaCl D M NaCl Test Solutios used i Experimet: 4-5 Solutios A through D are NaCl salt solutios of differet osmolarity. Distilled water (DH 2 O) is used as a cotrol for all treatmets ad is, by defiitio, hypotoic. Solutios E through H cotai a series of alcohols that differ i lipid solubility (i.e., how readily the alcohol passes through the membrae). Ethaol is the most lipid- E. 0.3 M ethaol (EtOH) i DH 2 O F. 0.8 M ethaol (EtOH) i DH 2 O G. 0.8 M ethylee glycol i DH 2 O H. 0.8 M glycerol i DH 2 O soluble (meaig that it ca easily diffuse through the membrae) ad glycerol is the least lipid-soluble. If the ormal osmolarity of the blood body is about Osm (Osm refers to the cocetratio of particles, so a Osm solutio has a particle cocetratio of M), what cocetratio of salt is isotoic to blood? (Remember that NaCl breaks up ito

6 Bio 26 Lab 4 Membraes 2 ios, Na + ad Cl -, whe dissolved). You should uderstad why the hemolysis rates differ as they do for these differet substaces, ad predict from a series of similar molecules which will have the highest hemolysis rates. Experimetal Procedure:. Usig a 0-ml glass pipette, trasfer 0 ml of each of eight test solutios ito separate test tubes. You should label the tubes (with tape ad/or a marker). A separate pipette should be used for each solutio you ca do this by placig a pipette i each of the solutio tubes o your bech. All solutios should be at room temperature. 2. Measure the time of hemolysis for oe solutio at a time, as follows. You will eed a clock/watch ad oe small square of Parafilm to seal each test tube. The measuremet is based o the time that it takes for hemolysis to progress to the poit where a black ad white image ca be see through a RBC suspesio i the test solutio. Each ru should be coducted quickly ad efficietly. If you have three people i your group, oe perso ca watch the clock, oe perso ca hold the test tube ad image, ad the third perso ca pipet the blood. 3. While holdig the test tube with parafilm at ready, use a P-200 pipettor to trasfer 50 μl (0.05 ml) of hepariized whole blood to the test tube. Whe pipettig blood, push ad release the pluger o the pipettor slowly so that the RBC's are ot ruptured by shear forces as they pass through the pipet tip. Start timig whe you add the blood - this is time-zero. 4. Immediately seal the tube with Parafilm. Mix it quickly by tiltig it upside dow oce. 5. Immediately hold the tube i frot of the black ad white image provided at your lab bech. Lookig at the image through the diluted blood i your test tube, watch for the poit whe the image o the film ca first be clearly resolved. This is the stop time. Record elapsed time, i secods, o your datasheet. If the image caot be discered withi te miutes, the record the hemolysis time as >600 sec. You should desig a table to record these values before comig to lab. 6. Repeat steps 4-6 for each test solutio. 7. Whe you are doe with the measuremets for all 8 solutios, discard the blood ad rise out all test tubes. 8. Copy your data oto the computer datasheet opeed by your TA. You will use data for your etire lab sectio for your aalysis. You recorded the time, i secods, that it took for the image to resolve through the RBC suspesio. Whe you aalyze the data (sectio IX below), you will take the reciprocal of time of hemolysis ad record that as the relative rate of peetratio i sec

7 Bio 26 Lab 4 Membraes VII. EXPRESSION AND INHIBITION OF RBC AQUAPORIN Aquaporis were oly recetly discovered (Presto et al. 992), first i red blood cells, ad later i water-permeable membraes of other tissues such as i kideys ad itesties. A commo model for observig the behavior of aquaporis is the oocyte (that is, a ufertilized egg) of the Africa Clawed Frog, Xeopus laevis. If mrna for the aquapori protei from huma RBC's is ijected ito a Xeopus oocyte, the oocyte will traslate this mrna ito the aquapori protei. The by a ukow mechaism the aquapori proteis isert themselves ito the oocyte membrae. I this experimet, we will attempt to observe the effect of huma aquapori expressio i Xeopus oocytes. Aquaporis ca be ihibited by certai compouds, such as mercury salts ad Tetraethylammoium Chloride (TEA). If aquaporis are importat i the movemet water across a oocyte membrae, how ca we use TEA as a cotrol i experimet? For this experimet, you will observe the rate at which the oocytes chage diameter, usig a ocular micrometer. Sice the oocytes are large (>mm i diameter), we will be usig dissectig microscopes. You will eed two microscopes per group. Whe addig the TEA ihibitor, you will icubate the eggs for at least 5 miutes prior to testig to make sure that TEA has boud to the aquaporis before the oocytes are added to the low-osmolarity test solutio. This experimet will be discussed further durig your lab sessio. Below is a summary of the experimetal desig. You will receive oocytes i a buffer called ND96, which is isoosmotic with oocytes ad has a osmolarity of 200 mosm. For the experimet, you will first choose a oocyte from the appropriate treatmet ad measure its diameter. The usig a pipet, trasfer the oocyte to a small Petri dish cotaiig a :3 dilutio of ND96 (2 parts water to part ND96) what is the osmolarity of this test solutio? After trasferrig the oocyte to the test solutio, measure two types of data: () oocyte diameter at 30 secod itervals (util 5 miutes or oocyte rupture, whichever comes first), ad (2) the time at which the oocyte ruptures. Treatmet #: Oocyte Experimetal Desig: Type of Oocyte used: TEA Icubatio? Ijected with 50µl water No 2 Ijected with 50µl RNA No 3 Ijected with 50µl RNA Yes 4-7

8 BIO 26: ENERGY FLOW IN BIOLOGICAL SYSTEMS VIII. DATA ANALYSIS - COMPARISON OF MEANS USING STANDARD ERROR Whe you have recorded your data, the class data will be pooled (i.e. the data set will iclude data collected by all studets i your lab). You should first covert each data poit to relative rate of peetratio i sec -, by takig the reciprocal of time to resolve the image o the slide. I order to preset ad iterpret these results, you will plot the rate of peetratio for each of the solutios used ad add stadard error bars for each mea. Stadard Error Bars To help uderstad the meaig ad use of stadard error bars, preted that you coducted a experimet o red blood cell hemolysis with three solutios that we'll desigate 'X' ad 'Y.' Assume that you coducted several trials (replicates) for each solutio usig the same methods as i our hemolysis experimet, measurig time to lysis ad calculatig relative rate of peetratio for each trial. The ext step is to fid the mea (or average) rate of peetratio for each solutio these meas will be compared to see if the solutios differed i terms of rate of peetratio. A statistical problem immediately presets itself, however. There may very well be differeces i the meas, but how differet must the meas be before we are cofidet that oe solutio (or treatmet) is differet from aother. (Note that there are two treatmets i this imagiary experimet solutio 'X' ad solutio 'Y'.) Scietists aswer this questio by comparig the differeces amog replicates withi treatmets to the differeces betwee treatmets. If the differeces betwee the treatmet meas are o greater tha the differece amog replicates i the same treatmet, the the treatmets themselves (i.e. the type of solutio to which red blood cells were added) had little or o effect blood cell hemolysis. To illustrate this, suppose that our hypothetical experimet had five replicates per treatmet (i.e., there were three tests for each solutio). Suppose that the mea rate of peetratio for solutio X is 0.65 sec -, ad the mea rate of peetratio for solutio Y is 0.46 sec -. Does this differece mea aythig? The oly way to tell is to look at the variatio withi the treatmets. We will examie two possible cases, which we ll call Case I ad Case II, each havig the same treatmet meas. The experimetal data for the two cases are show i Table 4.. Table 4.. Hypothetical data for hemolysis experimets, deoted Case I ad Case II. Case I Treatmet : Solutio X Treatmet 2: Solutio Y Case II Treatmet : Solutio X Treatmet 2: Solutio Y Mea: Mea: Note that treatmet meas are the same for both cases 4-8

9 Bio 26 Lab 4 Membraes I Case I there is a lot of variatio withi the treatmets (i.e., the idividual amouts eate from the three disks i each treatmet differ greatly from each other), whereas i Case II there is very little variatio withi treatmets. These relative differeces betwee the two cases are oly evidet after studyig the data closely what we really wat is a coveiet way to graphically represet these differeces. This is where the stadard error comes i: the stadard error of the mea is a statistical measure that is commoly used to graphically show variatio withi a treatmet. (I fact, the stadard error is oly oe of several statistics that measures the variatio withi a group of umbers, but it is the oe most commoly used o graphs.) We will discuss this statistic below the goig may be tough, but please stick it out ad read through the etire discussio. The stadard error of the mea (SE) is defied by the followig complicatedlookig formula: SE = = i = ( ) 2 x x i ( ) x i ) i =( 2 x i i = ( ) (2) 2 I the equatio, is the umber of replicates i the sample (3 i our hypothetical example), ad x represets the value for the parameter beig measured (e.g. peetratio rate). May electroic calculators have statistics fuctios ad will calculate the SE for you if you type i the idividual observatios; this method is highly recommeded. Note that though may calculators with statistics fuctios do ot calculate SE, they most likely do calculate stadard deviatio, or s. A simple coversio from stadard deviatio to stadard error is: SE = = = i = s i = ( ) 2 x x i ( ) ( ) 2 x x i where s = stadard deviatio calculated from the sample data. (3) To calculate the stadard error by had, it is easiest to use the right half of equatio (2) above. It is coveiet to set up a table with values for x ad x 2 for each replicate. Table 4.2 is such a table for case I of the hypothetical experimet o the previous page. 4-9

10 Table 4.2 Calculatio of stadard error for Case I Bio 26 Lab 4 Membraes Treatmet : Solutio X Treatmet 2: Solutio Y x x 2 x x Sum: For treatmet i Table 4.2, ( x i ) i= ad 2 = A large SE idicates much variatio, a small SE little variatio. I our hypothetical examples the SE s were: Table 4.3. Stadard Errors for Hypothetical Hemolysis Experimet Treatmet : Solutio X Treatmet 2: Solutio Y i= x i = Case I: Case II: Therefore, SE treatmet = = i= 0.3 ( x ) i 2 ( ) ( ) i= x 2 i ( 3.25) 2 (To be certai that you ca do so, use the data i Table 4. to calculate the SE s for Case II.) There are formal statistical techiques for usig SE s to determie whether differeces amog treatmet meas are sigificat. For the purposes of this lab, we will simply poit out that if the differece betwee meas is small relative to the SE, the the differece is ot meaigful. If the differece betwee the meas is large relative to the SE, the the differece is likely to be sigificat. For both hypothetical cases above, the differece betwee meas is 0.9; this is small relative to the SE s for case I, but it is large relative to the SE s i case II. Oly i case II would the experimet have show a potetially sigificat result. 4-0

11 Bio 26 Lab 4 Membraes Now we have the tools to create the graphical compariso that we ve bee workig towards. The compariso betwee meas relative to SE s ca be see at a glace whe data are preseted graphically, as show i Figure 4.4. I each case, the top of the large bar or box represets the mea for the treatmet. The SE is represeted by a error bar above ad below the mea the legth of each error bar is the value of the SE. Thus for treatmet of case I, the top of the large bar is positioed at 0.65; oe error bar exteds from the mea to (i.e., mea + SE = ), ad the other error bar exteds from the mea dow to (i.e., mea - SE = ). The visual compariso of sample meas is as follows: two meas are sigificatly differet if their correspodig stadard error bars do ot overlap. It is easy to see from the graph of case I that the error bars for the two treatmets overlap, ad that the differece betwee meas is mior compared to the SE we would coclude that i case I, the treatmet meas are ot sigificatly differet. For case II, the error bars do ot overlap ad the SE s are small compared to the differece betwee treatmet meas i this case the treatmet meas are most likely sigificatly differet.. Warig!! The Stadard Error Bar Method is a crude visual test Oe thig we eed to stress is that the stadard error bar method described above is a crude visual test. The visual compariso of meas does ot take the place of a statistical test. You ca use this visual test if you are i the audiece at scietific semiar or readig someoe else's paper - i these cases, you persoally do ot have the data ad caot do a statistical test. However, if you are presetig your ow data to the scietific commuity, you should perform the appropriate statistical test. The bottom lie here: whe you have the data, always use a statistical test to determie the sigificace of differeces betwee treatmets. I this lab we are usig this quick ad dirty method to avoid usig a set of complex statistical tests (i the hemolysis experimet, they would be Aalysis of Variace ad a set of post-hoc comparisos of meas), which are actually the more valid method of comparig meas. Whe we compare two meas usig stadard error bars ad fid that the error bars do ot overlap, our coclusio should be that the two meas are probably sigificatly differet to have a defiitive aswer we must compute the appropriate statistical tests. For the purposes of this lab, though, the visual test will suffice. Rate of Peetratio per sec Case I Case II Treatmet Treatmet 2 Leaf area eate (cm 2 ) Treatmet Treatmet 2 Figure 4.4 Meas ad stadard error bars for the example data. 4-

12 Bio 26 Lab 4 Membraes VIII. WRITTEN ASSIGNMENT You will use the whole class dataset for your lab assigmet, which will be i the Collab folder for your lab sectio. You are welcome to discuss the class data with your lab parters. However, you should work aloe whe makig the fial graphs ad writig aswers to the questios listed below. ASSIGNMENT CONTENTS A. Two graphs: RBC hemolysis ad Oocyte data. You should use the datasheet i Collab folder for your lab sectio. Ope the data spreadsheet with Microsoft Excel ad save it i your HOME folder you might wat to give it a differet ame. RBC graph: ). Covert all values for Rate of hemolysis to Relative Rate of Peetratio i.e., take the reciprocal of rate of hemolysis. 2). Calculate averages ad stadard errors for each of the test solutios 3). Create a bar graph with the differet solutios o the x-axis ad rate of peetratio o the y- axis. You should have Excel draw error bars for each bar o the graphs, with the size of each error bar equal to the stadard error. See the lik o the lab web page for help with plottig error bars. See also pages i Kisely's "A Studet Hadbook for Writig i Biology, 2 d ed." for help o usig Excel to graph data. The graph should have a complete captio. Oocyte graph: ). Calculate averages ad stadard errors for each of the three treatmets. B. Iterpret the data. 2). Create a bar graph with the oocyte treatmets o the x-axis ad rate of oocyte expasio o the y- axis. You should have Excel draw error bars for each bar o the graphs, with the size of each error bar equal to the stadard error. The graph should have a complete captio. The first step i iterpretig the data is decidig which bars o your bar graph seem to be sigificatly differet. The, aswer the questios below. Please type all aswers..) For the distilled water ad NaCl solutios, which seemed to be sigificatly differet? 2). How do you explai the apparetly sigificat differeces betwee the water ad salt solutios? 3). Were 0.45M NaCl ad 0.350M NaCl differet why or why ot? 4). How do you explai differeces betwee the differet alcohols? (Hit: Remember that alcohols vary i their ability to cross the membraes, ad water ad alcohol ca move across the membrae simultaeously.) 5). Were aquaporis expressed i the Xeopus oocytes? Support you aswer. 6). Did TEA seem to ihibit trasport of water by aquapori? Support you aswer. 4-2