one of key   topics in contemporary biology is a dynamic morphology of cells , and endothelial cells ( ec ) , in particular , which is closely related to cooperative dynamics of cytoskeleton and cell adhesive structures . cell systems participating in this process comprise not only structural components and associated mechanisms ( cytoskeleton fibrils , molecular motors , and adhesive receptors ) , but also regulatory and signaling elements modulating the dynamics and interactions among these structural units . the primary function of the ec lining the inner surface of all vessels is to regulate permeability of vascular walls and control the exchange between circulating blood and tissue fluids . cytoskeleton re - organization changes the cell shape and provides structural basis for an enhancement as well as a loss of endothelial barrier function . increases in vascular permeability , common for a number of human pathological states and diseases , such as inflammation , asthma , sepsis , acute lung injury , ischemia , and diabetes , can lead to severe , and even fatal , organ dysfunction     [ 16 ] . previous studies , published by us and by others , have proved that normal functioning of the endothelial barrier is provided by the balance between contracting and stretching forces generated by cytoskeleton proteins     [ 3 , 5 , 79 ] . moreover , endothelial cell - cell adherens junctions ( ajs ) , largely composed of vascular endothelial cadherin ( ve - cadherin ) , are the basic structure of endothelial permeability regulation because of their dynamic ability to open and close     [ 10 , 11 ] . the role of actin cytoskeleton in the formation , maintenance , and functionality of ajs in ec is well characterized     [ 1214 ] . however , the potential link between ajs and microtubules has not been well investigated . as a major component of cytoskeleton , microtubules have important functions in various cellular processes , such as cell shape formation , cell polarization , and motility     [ 1522 ] . in many cell types , microtubules are organized in a radial array with their minus - ends anchored at the centrosome and their plus - ends extending toward the cell periphery where they are involved in a number of essential cellular events     [ 2325 ] . microtubules are known to interact with the cell - cell adhesion machinery in fibroblasts      and in epithelial cells     [ 2731 ] . it was also shown that microtubule dynamics is an important factor in regulation of cell - cell contacts     [ 3032 ] . our previous data demonstrated the critical involvement of the microtubule disassembly in induced ec barrier dysfunction     [ 8 , 9 , 33 ] and allowed us to assume that microtubule dynamics is an early event in the circuit of the reactions leading to the changes in pulmonary ec barrier permeability . here we described a cellular model established in physiologically - relevant human pulmonary artery ec ( hpaec ) . this model is suitable for real - time study of microtubule organization and dynamics in the quiescent human ec monolayer as well as in the cells treated with the agents compromising / enhancing endothelial barrier . using this model we were able to obtain several key parameters of microtubule organization in the endothelium such as a ratio between stable and dynamic microtubule subpopulations , direct measurement of microtubule growth rates , and their difference in single ec and the cells grown as a monolayer . in our opinion , this cellular model would also allow us to study the involvement of microtubules in the barrier - protective / compromising mechanisms activated in pulmonary endothelium by various pharmacological agents of interest . ( usa ) and cultivated in complete egm-2 medium ( clonetics biowhittaker ) at 37c in an atmosphere of 5% co2 . experiments were performed on cultures at 610th passages , seeded at 30% confluence , and utilized either at 50% confluence or when fully confluent ( depending on the type of experiments ) . as primary antibodies for microtubule staining , we used monoclonal mouse antibody against -tubulin ( icn , usa ) ( 1  :  200 ) , monoclonal mouse antibody against acetylated tubulin ( accurate chemicals , usa ) ( 1  :  100 ) , and monoclonal mouse antibody against tyrosinated tubulin ( icn , usa ) ( 1  :  100 ) . antimouse antibodies conjugated to the fluorescent dyes alexa-488 or alexa-594 ( molecular probes , usa ) ( 1  :  100 ) were used as secondary antibodies .   prior to immunofluorescence staining , hpaec grown on glass coverslips were fixed for 10 minutes with a 1.5% solution of glutaraldehyde ( sigma , usa ) in phosphate - buffered saline ( pbs ) , ph 6.8 ( sigma , usa ) and washed 3 times with pbs ( each washing session lasted 10 min ) . fixed cells were permeabilized with 0.1% triton x-100 ( sigma , usa ) in pbs for 15 minutes and washed thrice with pbs for 10 minutes . to avoid background fluorescence , prior to staining with antibodies the cells were treated with a 0.2% solution of sodium borohydride ( nabh4 ) ( sigma , usa ) in pbs ( 10 min , three times ) and washed 3 times with pbs for 10 minutes . the next step included incubation of cells with primary ( 30 min ,   37c ) and secondary ( 30 min , 37c ) antibodies . the coverslips were mounted on slides in water / glycerol mixture ( 1  :  1 ) as priming medium . prior to assays , cover glass edges were sealed with nail polish for better fixation of the samples . immunofluorescence stainings of ec monolayers were examined under a nikon eclipse te2000 microscope ( nikon intech co. , japan ) supplied with a 60/1.4 objective . the most spread cells were selected in order to ensure better visualization of the cell structures . images were recorded using hamamatsu orca-2 ( hamamatsu photonics , japan ) digital cooled ccd camera supported with metaview software ( universal imaging , usa ) . image processing was performed using metamorph ( universal imaging , usa ) and adobe photoshop 7.0 ( adobe inc . quantitative analysis of microtubules was carried out as described previously and included measurement of their fluorescence using the metamorph software and analysis of digital images collected with a digital ccd camera     [ 8 , 9 ] . for the analysis , extended focus images of well - spread cells with minimal thickness were used . microtubule subpopulations in the area of interest were computed by the original image segmentation with threshold set to 200% of background level and by calculating the percentage of above - the - threshhold pixels . the relative area occupied by the microtubule network in different cell compartments was calculated in three different areas : ( 1 ) an area circumjacent to the cell periphery that was 5  m from the cell margin ; ( 2 ) an area circumjacent to the cell periphery that was 10  m from the cell margin ; and ( 3 ) the inner compartment , that is , the internal cytoplasm ( 10  m from the cell margin ) not including the first two areas . the ratio between the area occupied by microtubules to the measured area was determined separately for each measured area . statistical analysis with performed with sigma plot 7.1 ( spss science , usa ) and excel ( microsoft corp . sigma plot 7.1 software was used for graphical data presentation . to calculate the microtubule growth rate in living hpaec we used akhmanova [ erasmus university , rotterdam ] ) , which serves as a marker of growing distal tips ( plus - ends ) of microtubules     . usa ) was used for transfection of plasmids into hpaec cells according to the manufacturer 's protocol . transfected cells were selected for imaging by gfp fluorescence . for live imaging , the cells were cultured on glass - bottomed dishes with no.1s coverslips ( iwaki , japan ) . images of cells were collected with a pc - based deltavision optical sectioning system using planapo 100x/1.40 na oil or planapo 60x/1.40 na oil ph3 objectives ( olympus ) . images were acquired with a cooled ccd camera ( hamamatsu photonics ) with an appropriate nd filter , binning of pixels , exposure time , and time intervals . fluorescence signals were visualized using the endow gfp bandpass emission filter set ( 41017 , chroma ) for gfp imaging . quantitative analysis of the microtubule dynamics was carried out on time - lapse movies of cells expressing eb3-gfp . microtubule growth rates were obtained by tracking eb3-gfp comets at microtubule plus - ends ( 1 second / frame ) using imagej software linked to an excel spreadsheet . previously , we demonstrated that the microtubule population in ec is heterogeneous and partly represented by posttranslationally modified ( acetylated ) microtubules     . acetylated microtubules are less dynamic than intact tyrosinated microtubules and more resistant to the effects of external factors . therefore , under conditions compromising vascular endothelium integrity , they may confer stability on the endothelial microtubule network . moreover , we can assume that some ec barrier - enhancing factors may shift the ratio in favor of stable microtubule subpopulation and increase overall stability of the ec cytoskeleton . since the ratio of dynamic ( tyrosinated ) and stable ( acetylated ) microtubules in ec has not yet been determined , in first set of experiments , we addressed this question using immunofluorescence staining of the modified tubulins followed by visualization and quantification of dynamic and stable subpopulations of microtubules . in our study , we also focused on direct tracking growing distal tips of microtubules ( plus - ends ) in order to establish an adequate model to study microtubule dynamics in ec monolayers and single cells . we also wanted insights for further analysis of microtubule - dependent response in ec under barrier- compromising / enhancing conditions . immunofluorescence staining of -tubulin in human ec revealed that the microtubule network had a well - defined convergent center near the nucleus ( figure 1(a ) ) . the density of the microtubule network was the highest in the internal cytoplasm , but diminished gradually in the direction of the cell margin . single microtubules were visualized at the cell periphery . on average , microtubules occupied 47.9  4.1% of the total cell area ( n = 20 ) ( figure 1(d ) ) . in the cell interior , the relative area occupied by microtubules was 83.3  5.8% ( n = 20 ) . the microtubule population density decreased from the cell center to cell margin . in the area circumjacent to the cell periphery ( 10  m from the cell center ) , microtubules occupied up to   28.3  7.4% of the total cell area . at the cell edge ( 5  m from the cell margin ) , microtubules occupied   15.7  6.6% of the cytoplasm area . as mentioned above , endothelial microtubule network is heterogeneous , which is consistent with the dynamic characteristics of its constituent microtubules . the microtubule population can be divided into two subpopulations : stable , modified ( acetylated ) , and dynamic ( tyrosinated ) microtubules . the subpopulation of stable ( acetylated ) microtubules was identified by immunofluorescence staining using antibody against acetylated tubulin . in quiescent ec monolayers , acetylated microtubules were predominantly localized in the cell center and were devoid of a well - defined convergence center ( figure 1(b ) ) . quantitative analysis demonstrated that acetylated microtubules occupied only 17.1  4.3% ( n = 20 ) of the cell surface or   35% of the total microtubule network area ( figure 1(d ) ) . the subpopulation of intact tyrosinated microtubules   was identified by immunofluorescence staining of ec using antibody against tyrosinated tubulin ( figure 1(c ) ) . the distribution pattern of tyrosinated microtubules was similar to that of the microtubule network immunostained with anti--tubulin antibody ( figures 1(a ) and 1(c ) ) . tyrosinated microtubules ( n = 20 ) occupied 47.0  3.6% of the total cell area , which is consistent with the corresponding parameter for the microtubule system in general ( figure 1(d ) ) . in addition to quantitative analysis of the areas occupied by dynamic and stable microtubules in different compartments of human ec , we calculated microtubule plus - ends growth rates in ec . first , high microtubule density at the centrosome makes it difficult to visualize individual microtubules . second , the ec are generally thick in the region of the centrosome , which results in substantial out - of - focus fluorescence that degrades imaging of individual fluorescently labeled microtubules . to specifically visualize microtubule growing ends , we used eb3-gfp as a marker of growing distal tips of microtubules     . microtubule growth rates were obtained by tracking eb3-gfp comets at microtubule plus - ends . we evaluated microtubule growth rates in the central region and near the cell periphery of human ec by tracking individual eb3-gfp dashes as they emerged from the centrosome and grew outwards in the direction of cell periphery ( figure 2 ) . in hpaec monolayer , the instantaneous rate of microtubule plus - ends growth was the highest at the cell center , in the centrosome region , and it was 25% lower near the cell margin ( figure 3 ; table 2 ) . we observed plus - ends growth episodes at a rate exceeded 40  m / min in the centrosome region but not on the cell periphery . a portion of microtubule plus - ends grew persistently from the centrosome , reaching the cell edge before the comet of eb3-gfp was lost . in some cases , gfp - labeled growing plus - ends were observed to reach the edge of the cell and continue to grow along the edge . this observation suggests that the cell edge in human ec does not always alter the behavior of the microtubule end , and , at least in some cases , the plus - end can change its trajectory and continue to grow along the cell border . analysis   of growth rates for radially - oriented microtubules and microtubules growing along the cell border showed very similar instantaneous rates of microtubule plus - ends12.4  0.1  m / min ( n = 206 ) and 11.9  0.1  m / min ( n = 115 ) , respectively . we examined microtubule dynamics in the centrosome region in cells contacted on all sides with neighboring cells ( ec monolayer ) and in single cells . the results obtained using ec expressing eb3-gfp visually demonstrated that growing microtubule plus - ends were distributed in a relatively uniform manner in a single human ec as well as in a cell in monolayer where cell - cell contacts , mainly ajs , were formed and well - organized . quantification of microtubule plus - ends growth rates showed that in single cells the instantaneous rate of microtubule plus ends growing in the centrosome region was 20.6  0.6  m / min ( n = 72 ) . comparing this instantaneous rate with that calculated for the monolayer , we concluded that in monolayer - growing cells , the rate of microtubule plus ends growth was 20% lower than in single cell ( figures   2 , 3 ; table 2 ) . the histogram of centrosomal microtubules growth rate distribution demonstrated two peaks in single human ec : the first peak showed a correlation with a growth rate near 1820  m / min , the second peak of the histogram started with a growth rate more than 32  m / min and   frequency of episodes of fast growth that had a second maximum at 4550  m / min . it is possible to explain an origin of the second peak on the microtubule growth rates histogram ( figure 4 ) , based on two assumptions . first , one can assume the existence of two different groups of centrosomal microtubules with various growth rates . from the other side , it is possible that the same individual microtubule can grow quickly for a few frames of the recording and then grow more slowly . however , the analysis of the entire data file of microtubule growth rates measurement ( data is not shown ) has not revealed sudden fluctuations in growth rates of individual microtubules depending on observation time . the presented representative examples of microtubules plus - ends displacements ( figures 2(f ) , 2(g ) , 2(h ) , and 2(i ) ) demonstrate that growth rates of individual microtubules does not undergo dramatic changes during observation time . therefore , based on data obtained , we could distinguish two different groups of centrosomal microtubules according to their growth rates . most of microtubules ( 85% of growth episodes  figure 4 ) belonged to the group with the growth rates in the first group coincided with those characteristic for microtubules in the ec monolayer ( average growth rate 14.4  3.3  m / min ) . average growth rate in the second (  fast  ) group was much higher59.4  4.3  m / min . since growth rate of  fast  microtubules was approximately three times higher , their contribution to average growth rate was quite significant . our results suggest that in the population of single cells , the average growth rate was about 20% higher than the growth rate of microtubules polymerizing in the ec monolayer .   by analyzing the local microtubule dynamics in the internal cytoplasm and near the cell margin in human ec , we have found that the microtubule plus - ends growth rate on the cell periphery ( near the cell margin ) is lower than in the internal cytoplasm . we could also conclude that the growth rate on the periphery did   not depend on microtubule tips growth orientation along or perpendicular to the cell border . we can speculate that microtubule growth rate closely depended on ajs availability , and it is lower in monolayer - growing ec where cell - cell contacts were fully formed . our results demonstrate ( figure 1 ) that microtubules are highly dynamic structures in human ec . their nonstatic plus - ends are located near the cell edge , whereas acetylated stable microtubules ( ~1/3 of dynamic microtubules population ) are found exclusively in the central part of the cell and their plus - ends are distant from the cell edge ( table 1 ) . these data suggest that exactly the same dynamic microtubules may interact with the cell - cell adhesion machinery in human ec , as it was shown previously for fibroblasts      and epithelial cells , where microtubules are responsible for the biogenesis and turnover of the cell junctions     [ 2731 ] . similarly to those cell types , microtubule dynamics may be involved in the regulation of cell - cell ajs to control ec specific functions , in particular , barrier permeability maintenance . however , to prove this hypothesis , we will need to perform special studies using the substances affecting the integrity of the endothelial monolayer and our ec model expressing eb3-gfp . hpaec expressing the gfp fusion protein specific for microtubule plus - ends were indispensable in acquiring the parameters of microtubule dynamics in real time . we were able to measure microtubule growth in the internal cytoplasm , near the centrosome , and at the periphery of human ec ( figure 2 ) . microtubule growth rates determined using eb3-gfp ( or some others ) plus - end tracking proteins are higher than those measured using fluorescein - labeled tubulin     [ 36 , 37 ] . eb3 was shown to present at microtubule plus - ends during growth phases but disappeared within 5 seconds after the transition from growth to pause or shortening phase . slight pauses in the microtubule growth may be included in the measurement of microtubule growth rate calculated using fluorescein - labeled tubulin approach , thus reducing the apparent growth rate . therefore , to avoid any discrepancy , we compared our results with published data obtained using similar approach only . dynamic instability behavior of microtubules is cell - type - specific     [ 3844 ] and growth rates are generally dissimilar in different cell types and in different cell areas . in hippocampal culture , the average velocity of eb3-gfp ( 13  m / min ) is no different in the cell body , the neuritis , or in the growth cone , but it is twice as high in the glia and in cos-1 cells     . however , early studies of microtubule behavior in living cells were limited in the cell interior but not near the cell periphery , where individual microtubule ends could be detected and their dynamics could be quantified . more recently , it has become clear that a complete understanding of microtubule behavior requires knowledge of events in the central cytoplasm and in the internal cytoplasm     [ 36 , 45 ] . our data of direct quantification of microtubule dynamics in ec , obtained for the first time , clearly demonstrated ( figure 3 ) that the growth rates in ec are comparable with those obtained for epithelial cells and fibroblasts     [ 36 , 37 ] . indeed , quantitative analysis of microtubule dynamics showed that in the case of eb3-gfp ( or another plus - end tracking proteins ) labeling the instantaneous rates measured in the internal cytoplasm of cho fibroblasts was about 16 - 17  m / min . this was similar to that which we calculated for hpaec ( figure 3 , table 2 ) . unexpectedly , our data are comparable with the rates determined for epithelial llcpk1 cells and for cho fibroblasts polarized and migrating directionally into the wound . the instantaneous rate of microtubule growth was similar for microtubules extending toward the front and rear of the cell : in llcpk1 cells the average growth rates were 17.9  7.7  m / min in the leading edge and 19.0  8.8  m / min the trailing edge , in cho cells16.0  6.8 and 17.2  7.7  m / min , respectively . thus , microtubule plus - ends are highly dynamic in the internal cytoplasm of ec , and their growth rate is comparable with microtubule growth rate in fibroblasts or in functionally active cytoplasm areas in polarized and migrating cells . the data we obtained ( figure 3 ) showed that the microtubule plus - end growth rate was reduced from the cell center to the cell periphery , indicating that the microtubule dynamics varied in different regions of human ec . on the other hand , microtubule growth rate was lower in ec cultivated in the monolayer than in single cells ( figure 4 ) , and it may well be that microtubule plus - ends growth rate decreased with ec monolayer formation . study of microtubule dynamics in living newt lung epithelial cells showed that microtubules in the extending lamellae at the leading edge are dynamic , whereas microtubules in lamellae that contact neighboring cells can be either dynamic or stable . our data ( figure 4 ) make it clear that in the centrosome region there are similar peculiarities in microtubule plus - ends behavior . it was shown that in polarized , motile cells , microtubules extended into newly formed protrusions at the leading edge . these  pioneering microtubules   demonstrated different behavior when compared with microtubules in nonleading , lateral edges , indicating the region - specific differences in microtubule dynamics . fast  episodes of growth which we detected in the centrosome region of single ec ( figure 4(b ) ) are analogues of such  pioneering  microtubules . therefore , microtubule plus - ends behavior is different in the centrosome region and on the periphery of ec . taken together , our results suggest the existence of different mechanisms of local regulation of microtubule plus ends elongation in ec . the microtubule cytoskeleton is a major determinant of cortical dynamics and microtubules can interact with the cortices of animal cells in a variety of ways . one such interaction involves microtubule plus - ends , which are commonly oriented towards the cell periphery     [ 25 , 48 ] . because of dynamic instability allows these plus ends may grow outwards and potentially explore peripheral structures , including integrin - based focal adhesions   and organize vesicular transport to the cell surface , as well as the delivery of regulatory molecules to the cortex . our findings suggest that microtubules in human ec are dynamic , with high growth rate comparable to microtubule growth rate in fibroblasts in the cell interior and lower growth rate near the cell boundary in the area of cell contacts ( figure 4 ) . we suggest that high microtubule dynamics and local distinctions in microtubule growth rate underlie the specific function of ec where fast delivery of molecular signals to the cell edge ( to the area of cell - cell junctions ) is urgent for their active and fast local regulation in response to external and internal signals . rapid growth allows nascent microtubules to elongate from the centrosome area to the cell boundary in a very short time . potential mechanism of high growth rate of these microtubules can be realized via their selective and more stable binding to the plus - end protein(s ) . this efficient association with the plus - end protein(s ) may support an efficient capture of tubulin molecules or enhancing lateral interactions between individual protofilaments . the proteins of eb family ( eb1 and eb3 ) may be involved in this process , since they control persistent microtubule growth and possess anticatastrophe activity in the cell   . in the ec monolayer , where aj contacts are organized , microtubules may interact with ajs and this interaction may lead to their stabilization in the area of the contact . cell - cell contact in lung epithelial cells was reported to stabilize the dynamic behavior of microtubule plus - ends     [ 32 , 46 ] . according to our results , dynamic microtubules are capable of adjusting existing contacts and can adjust endothelial permeability .
to understand how microtubules contribute to the dynamic reorganization of the endothelial cell ( ec ) cytoskeleton , we established an ec model expressing eb3-gfp , a protein that marks microtubule plus - ends . using this model , we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human ec and in the ec monolayer . 
 we demonstrate that the majority of microtubules in ec are dynamic , the growth rate of their plus - ends is highest in the internal cytoplasm , in the region of the centrosome . growth rate of microtubule plus - ends decreases from the cell center toward the periphery . 
 our data suggest the existing mechanism(s ) of local regulation of microtubule plus - ends growth in ec . 
 microtubule growth rate in the internal cytoplasm of ec in the monolayer is lower than that of single ec suggesting the regulatory effect of cell - cell contacts . 
 centrosomal microtubule growth rate distribution in single ec indicated the presence of two subpopulations of microtubules with 
 normal 
 ( similar to those in monolayer ec ) and  fast  ( three times as much ) growth rates . 
 our results indicate functional interactions between cell - cell contacts and microtubules .