escherichia coli cells are able to respond to change in environmental chemo - effector concentrations through the reversal of their flagellar motors .
attractants ( such as aspartate and serine ) promote counterclockwise rotation of the flagella , resulting in smooth swimming , whereas repellents ( such as phenol and ni ) promote clockwise rotation , resulting in tumbling .
these responses are mediated by membrane - bound , methyl - accepting chemoreceptor proteins ( mcps ) .
immunoelectron microscopy revealed that mcp - chew - chea complexes are clustered in vivo , predominantly at the cell poles , and merely weaker lateral clusters were observed .
it has been expected that the polar - localization changes according to environmental conditions , whereas there is no evidence showing the dynamics of localization - change have been reported .
conventional group - based experiments do not allow measuring the process of mcps clustering and its change in consecutive generations in individual cells , which is essential to estimate the change occurring during the alternation of generations . in order to understand epigenetic processes such as adaptation and selection ,
both the protein - dynamics and the cell - dynamics of particular single cells should be observed continuously for generations .
previously , we have developed the on - chip culture system exploiting the microfabrication technique and optical trapping [ 6 - 8 ] .
the system enabled us to keep the condition around the cells constant by exchanging the fresh medium continuously and by controlling the cell number with optical trapping .
individual cells swimming in microchambers were observed with a spatial resolution of 0.2 m by phase - contrast / fluorescence microscopy . to measure the localization - dynamics of expressed proteins and the motility of single cells
simultaneously , we used the on - chip microcultivation system and assayed intracellular proteins tagged with green fluorescent protein ( gfp ) . in this paper , we report the time course of the motility and the localization of the aspartate receptor protein ( tar ) for several cell generations .
we also report cellular responses caused by the addition and removal of the aspartate during cultivation .
figure 1a shows the top - view of the wheel - shaped - microchamber used in this experiment .
first , a single bacterium was placed in a microchamber and was isolated in the wheel region to swim along the track by sealing of semi - permiable membrane lid onto the microchamber .
then , the bacterium running around the circle structure was observed continuously by measuring the tumbling frequency and the protein - localization dynamics . when the cell divided into two daughter cells , one of these two daughter cells was picked up by optical tweezers , transported to the axle area and was held in this region with optical tweezers continuously to stop it growing .
the microchambers were etched into a 0.2-mm - thick biotin - coated glass slide , and were covered with a semi - permeable avidin - coated cellulose membrane lid ( pore size : molecular weight -25,000 ) to separate the microchambers from the medium - buffer - exchange region .
the cultivation buffer in the microchamber was continuously refreshed by diffusion through the membrane ( refresh rate  approximately one minute ) to maintain the homogeneous medium environment .
a bacterium was gave chemical stimulation by changing contents of medium supplied from the medium - buffer - exchange region .
( b ) the magnified micrographs show sensor - protein dynamics of isolated single e. coli at times of ( a ) 0 min , ( b ) 180 min , ( c ) 225 min , ( d ) 315 min , and ( e ) 435 min after the inoculation .
we measured tumbling frequency and tar - localization ratio of isolated e. coli in m9 minimal medium containing non - attractor amino acid ( asn , arg , cys , ile , gln , thr , thy , trp , val , his , phe , pro , met , lys , and leu ) at 1 mm and 0.2% glucose at 30c ( fig .
2 ) . to visualize tar protein in living e. coli cells , tar was tagged by green fluorescence protein in aw539 strain .
the tar - localization ratio is estimated by measuring the ratio of the cytoplasm fluorescence intensity and the polar fluorescence intensities .
time course of tumbling frequency ( blue circles ) and tar - localization dynamics ( red squares ) with chemical stimulation .
when the cultivation started , the tar - localization ratio ( red square ) was 2.5 and the tumbling - frequency ( blue circle ) was 0.5 ( s-1 ) ( fig .
after the 2nd cell division was occurred , minimal medium containing 1 mm aspartate was given to the 3rd generation of cell ( 135 min after microcultivation ) .
after adding the attractant , tumbling - frequency was decreased immediately compared to the previous generation .
localization of the aspartate sensitive sensor protein at two poles in escherichia coli also decreased quickly by half 45 min following medium change ( fig .
then , the aspartate was removed from the cultivation medium and cells were cultivated further to measure the recovery of tar - locarization dynamics ( fig .
, it took more than three generations to recover original pattern of tar localization ( fig .
this may indicate that the formation of tar - localization requires more time than its diffusion .
such an asymmetric reversibility of protein localization may contribute to the inheritance of the cells ' phenomenon caused by environmental change .
it also suggests a possibility that change of tar localization can be inherited by descendant cells and this can affect their motility and therefore their phenotype . in conclusion , we reported the following two topics :  a novel assay for observing the protein - localization dynamics and the motility for generations was developed .  the decreasing and recovery of the tar - localization in a living bacterium was monitored under environmental change .
this assay can potentially be used for measuring cell - dynamics and inheritance of those information from generation to generation with focusing each individual cell caused by the environmental change .
ii carried out the microchamber design , cell preparation , single cell observation , image analysis and drafted the manuscript .
 measurement of the correlation between sensor - protein expression , motility and environmental change is important for understanding the adaptation process of cells during their change of generation . 
 we have developed a novel assay exploiting the on - chip cultivation system , which enabled us to observe the change of the localization of expressed sensor - protein and the motility for generations . 
 localization of the aspartate sensitive sensor protein at two poles in escherichia coli decreased quickly after the aspartate was added into the cultivation medium . 
 however , it took more than three generations for recovering the localization after the removal of aspartate from the medium . 
 moreover , the tumbling frequency was strongly related to the localization of the sensor protein in a cell . 
 the results indicate that the change of the spatial localization of sensor protein , which was inherited for more than three generations , may contribute to cells , motility as the inheritable information . 
Finding
Authors' contributions