s2fieldsofstudy: Biology Medicine

text:
A single vaccination with an inactivated bovine respiratory syncytial virus vaccine primes the cellular immune response in calves with maternal antibody

Background The efficacy of a single dose of an inactivated bovine respiratory syncytial virus (BRSV) - Parainfluenaza type 3 (PI3) - Mannheimia haemolytica (Mh) combination vaccine, in calves positive for maternal antibodies, was established in a BRSV infection study. Results As expected the single vaccination did not have any effect on the decline of BRSV-specific neutralising or ELISA antibody. The cellular immune system was however primed by the vaccination. In the vaccinated group virus excretion with nasal discharge was reduced, less virus could be re-isolated from lung tissues and the lungs were less affected. Conclusions These results indicate that a single vaccination with an inactivated BRSV vaccine was able to break through the maternal immunity and induce partial protection in very young calves. It can be speculated that the level and duration of protection will improve after the second dose of vaccine is administered. A two-dose basic vaccination schedule is recommended under field conditions.

Background
The bovine respiratory syncytial virus (BRSV) has been established as an important viral component in the bovine respiratory disease complex (BRDC) [1]. The virus can act synergistically with other viruses such as the bovine parainfluenza type 3 virus (PI3) or bacteria such as Mannheimia haemolytica (Mh), Pasteurella multocida, Haemophilus somnus and Mycoplasma bovis to cause pneumonia [2]. BRSV also fulfils the Koch's postulates to be recognised as an aetiological agent of pneumonia in calves. Respiratory disease is however often milder under laboratory conditions than under field conditions [3][4][5][6].
Outbreaks of BRSV associated pneumonia typically recur every year [7] and most farms are affected. The mechanisms for the persistence of the virus in a farm are not well understood [8]. Based on sequence divergence among BRSV isolated during recurrent outbreaks in closed herds, (re)introduction of the virus into the herd prior to each new outbreak is the most likely explanation [9]. Airborne transmission of BRSV has been demonstrated under experimental conditions [10]. Different approaches have been followed to develop safe and efficacious BRSV vaccines including inactivated vaccines [11,12], (genetically) modified live vaccines [11,13], subunit vaccines [14], DNA vaccines [15] and vector vaccines [16,17]. Today inactivated and modified live BRSV vaccines are commercially available, most of them as combination vaccine with other antigens related to the BRDC.
The basic vaccination schedule for inactivated BRSV vaccines consist of two doses of 5 ml each, applied by subcutaneous injections, at an interval of 3 to 4 weeks. Remarkably most cases of severe BRSV are observed in calves aged 1-3 months, at which time they still possess maternal antibodies [18]. In order to provide efficacious protection against BRSV at this very young age it is essential that vaccines are able to overcome the interference of maternal antibodies and confer protection shortly after vaccination. Due to the high risk of BRSV associated pneumonia in the very young age group, it is also of great value to evaluate the level of immunity induced by the first vaccination before completion of the full primary course has taken place. The inactivated BRSV-PI3-Mh combination vaccine used in the study reported here has previously been shown to reduce the severity of clinical signs and virus shedding in two week-old MDA (+) calves that had received a primary course of two vaccinations four weeks apart. The calves were challenged with BRSV 3 weeks after the second vaccination. Moreover, in a previous study with the same vaccine, the efficacy in terms of antibody response and clinical protection has been established after application of a single dose of the vaccine to 4-5 week old calves with maternal antibodies [19]. In the study reported here two week old calves with maternal antibodies were vaccinated with a single dose, antibody levels as well as cellular immunity was measured. As it was expected that the maternal antibodies would interfere with the induction of clinical signs, lung pathology was used as a parameter to evaluate protection.

Animals
Ten Holstein crossbred calves were included in the study. They were removed from the dam directly after birth in order to prevent infection with BRSV or other pathogens. The calves were fed pre-collected colostrums with high antibody titres against BRSV in order to obtain similar antibody titres in all calves. The animals were identified by means of numbered ear tags. They were ranked by age and then allocated, on an alternating basis, to one of the two treatment groups in order to obtain the same average age for both groups. The complete study was undertaken in a blinded manner.
From the week before vaccination and onwards the calves were housed, separated by group, in two different rooms of an isolation unit. Strict barrier conditions between the two groups were maintained by means of individual filtered air supply to each room, sealed doors and by staff changing cloths and showering between the rooms.
One calf allocated to the control group had to be euthanised because of a persistent inflammation of the carpal joint before challenge.
All procedures were approved upfront by the Animal Care and Use Committee according to the Dutch animal welfare regulations.

Vaccination
A commercial batch of an inactivated combined BRSV, PI3 and Mh vaccine with alhydrogel and Quil A as adjuvants (Bovilis Bovipast RSP, Intervet/Schering-Plough Animal Health) was used. Five calves were given a single dose of 5 ml by the subcutaneous route into the neck. A second group of 5 calves was not vaccinated. The average age of the calves at the time of vaccination was two weeks.

Challenge
Four weeks after they had been vaccinated, all calves were challenged by aerosolisation with a wild-type strain of BRSV (CA-1, kindly provided by L. Gershwin) as described earlier [13]. The challenge dose was 10 5.9 TCID 50 in a 10 ml volume.

Assessment of clinical signs
From two days prior to challenge until the end of the study the calves were observed daily for clinical reactions and the body temperature was recorded. Special attention was paid to signs of respiratory disease such as coughing -spontaneous and stimulated (by palpation of the larynx), nasal or ocular discharge and respiratory rate.

Serology
To determine the calves' antibody response to vaccination and challenge, blood samples were taken at vaccination (day 0), just before challenge (day 28) and one week later (day 35). Serum samples were assayed for BRSV specific antibody by serum neutralising (VN) antibody and ELISA antibody.
The VN test was performed according to the end point dilution method in a microtitre format. Two-fold serial dilutions of the serum samples were incubated with a fixed amount of BRSV prior to the addition of Vero cells. After 5-7 days of culture, the plates were read for BRSV specific cytopathic effects. The VN titres were the reciprocal value of the highest dilution in which complete neutralisation of the virus was obtained. They were expressed as log 2 .
An in-house BRSV blocking ELISA was performed. Plates were first coated with a BRSV specific monoclonal antibody and secondly with BRSV antigen. After appropriate washing steps, dilution series of the sera were incubated followed by the incubation of a second biotinylated monoclonal antibody. Avidin-peroxidase complexes were allowed to bind to the biotin. A standard colour reaction using 3,3';5,5'tetramethylbenzidine as substrate was performed to quantitate the amount of bound second antibody. Antibody titres were calculated from the blocking percentage.

Cellular immune response
A lymphocyte activation assay was performed on heparinised blood samples taken at days 0, 28 and 35. In 24well plates heparinised blood samples (sample volume: 1 ml) were stimulated with Concanavalin A (Sigma Aldrich, 100 μl/ml blood) or lysate of BRSV infected MDBK cells. Negative control samples were treated similarly with culture medium or lysate of uninfected MDBK cells. After two days of incubation at 37°C Interferon γ (IFN γ) production was measured using a commercial ELISA (Bovine IFN γ EASIA, Biosource Europe S.A.) according to the manufacturer's recommendations.

Nasal shedding of challenge virus
From two days prior to challenge until the end of the study nasal swab samples were taken daily from all calves by means of swab sticks containing a sterile cotton plug. Separate swabs were used for each animal. The swabs were gently rotated up and down the sides of both nasal cavities several times. The swabs were then inserted into a tube containing 1 ml of sterile phosphate buffered saline medium. The liquid was transferred into suitable containers and stored at temperatures below -70°C until tested.

Post mortem examination
At seven or 10 days after challenge the calves were sacrificed. The lungs were inspected macroscopically and representative samples of organs showing any changes were taken. The samples were fixed with formalin and then processed into paraffin embedded sections. These were stained with haematoxylin and eosin and investigated microscopically.
Additional samples for virus isolation were taken from nasopharyngeal mucosa, tonsils, submandibular lymph nodes, each lung lobe and mediastinal and bronchial lymph nodes.
Tracheobroncheal lung washes were performed as follows. Both lungs were excised and flushed with 300 ml of saline. The liquid was recovered and stored at temperatures below -70°C until tested.

Titration of nasal swab samples and lung wash fluid for BRSV
The BRSV titre in nasal swab samples and lung wash fluid was determined according to the end point dilution method in a microtitre format. Five-fold serial dilutions of the samples were incubated on bovine embryo lung cells. Infection was evaluated by the development of BRSV specific cytopathic effects after 5-7 days of culture. Each sample was tested in quadruplicates.

Testing of tissue samples for BRSV
Homogenized samples were incubated in four-fold on a monolayer of Vero cells in 24-well plates. After 5-7 days of culture supernatants of the cultures were transferred onto fresh monolayers of Vero cells. The monolayers after the first and the second passage were read for BRSV specific cytopathic effects.

Statistical evaluation
The results were summarised using frequency tables. The Wilcoxon two-sample test was performed to compare: maximum titres in nasal swab samples, days of virus excretion and extinction values in the INFγ ELISA.
The above statistical tests were performed at 5% level of significance. The data analysis was done using SAS

Clinical signs and body temperatures
Only minor clinical signs were observed. There were no increased respiratory rates; in fact the only clinical sign of respiratory disease was coughing of individual animals. One vaccinated animal showed stimulated coughing, during examination, at 6 and 7 days after challenge. Another vaccinated and one unvaccinated animal coughed spontaneously on days 8 through 10.
Throughout the experiment none of the calves developed a fever (body temperature above 39.5°C).

BRSV specific immune reaction BRSV specific antibody response
All calves had moderate to high antibody titres at the start of the study (individual data not shown). The average VN and ELISA antibody titres at the time of vaccination in the vaccinated group were 9.6 log 2 and 8.2 log 2 (see table 1). These values are very similar to the average titres measured at the same time for the control animals (9.3 log 2 and 8.3 log 2 for VN and ELISA respectively). At the time of challenge (t = 28 days) and one week later (t = 35 days) the titres were at the same level or had dropped slightly for both groups.

BRSV specific cellular immune response
No cellular immune response, as determined in the lymphocyte stimulation assay, was detected at the time of vaccination (see table 2). At the time of challenge one out of five vaccinated animals were positive, all unvaccinated animals were negative.
One week after the challenge IFN γ production was measured in stimulated blood samples of four out of five vaccinated and one out of four unvaccinated control calves. The difference between the two groups was statistically significant (p < 0.05, Wilcoxon two-sample test).

Virus re-isolation from nasal swab samples
From day 4 to day 7 after infection BRSV could be reisolated from nasal swab samples from three out of four unvaccinated animals. (table 3). Only two out of five vaccinated animals shed detectable but low levels of

Gross and microscopic pathology findings
Macroscopic pathological findings differed substantially between the animals No lung lesions were observed in the lungs of two vaccinated and one unvaccinated animal. Only minor lesions were present in the lungs of two vaccinated and two unvaccinated animals. The lung of the fifth vaccinated animal showed dark red consolidation of 10-25% of the medial and apical lung lobes. In contrast all lung lobes of the fourth unvaccinated animal were affected and 50% of the right apical lobe and 80% of the medial lobe were consolidated.
BRSV non-specific microscopic lesions were present in two vaccinated and three control animals. Syncytial cells, which are characteristic for BRSV infection, were present in two vaccinated animals and suspected in one control animal.

Isolation of BRSV from tissue samples and lung wash fluid
BRSV could be re-isolated from tissue samples out of the respiratory tract in four out of five vaccinated and all unvaccinated animals (see table 4). The number of positive samples was higher in the unvaccinated group (average 4.5) than in the vaccinated (2.0).
The lung wash fluid of one vaccinated and one unvaccinated animal, both killed 7 days after infection, was found positive for BRSV. All other samples were negative.

Discussion
The clinical signs observed after intranasal challenge with virulent BRSV were rather mild in the control animals and therefore no conclusion on clinical protection can be drawn from this study. The difficulty of reproducing BRSV associated disease under experimental conditions has been reported previously [3][4][5][6]. In the study reported here calves with maternally derived antibodies were used and the challenge was performed as early as 6 weeks of age. At that point maternal antibody levels were still moderate to high. These maternal antibodies are likely to interfere with the BRSV infection as reported by Kimman and colleagues [18]. For this reason BRSV vaccination-challenge studies reported in the literature were often either performed in older animals [19,20] or colostrum deprived calves [21]. or the n.s. n.s.  challenge was performed later after vaccination [22,23], alternatively a combination of these different approaches was applied. However an early challenge of young calves with moderate to high maternally derived immunity, as described in this study, reflects the field situation. Under these conditions it is very difficult but also very important for a BRSV vaccine to provide protection. The vaccinated animals did not develop clinical signs other than provoked or spontaneous coughing in one animal. This observation is noteworthy since vaccine induced exacerbation of BRSV infections has been reported from field observations [24] and reproduced in the laboratory [25]. The pathophysiology of the disease in cattle was related to formalin inactivated BRSV. This is analogous to reports on human RSV enhancement after vaccination with formalin-inactivated RSV vaccine. The hypothesis is however not in line with the results of Mohanty and co-workers [26]. The vaccine used in this study also contained formalin-inactivated BRSV. The number of animals used here might be too low to allow general conclusions, however our results confirm earlier findings from laboratory studies [12,19] and unpublished field data that the vaccine does not enhance BRSV disease severity in naturally infected cattle.
In this experiment a single dose of the inactivated BRSV-PI3-Mh combination vaccine did not have any effect on the decline of BRSV-specific neutralising or ELISA antibody. This result was expected and is in line with other reports [19,20]. The role of different types of antibodies in protection against BRSV has been discussed in the literature [27]. Therefore, in this study, VN titres and ELISA titres were determined. However both types of antibody titres, VN and ELISA, followed the same profile e.g. animals with high VN titres also had high ELISA titres and vice versa. This finding indicates that the colostrums used to feed the calves were obtained from mothers with a similar history of BRSV vaccination and/or infection.
Earlier studies with the same vaccine have shown a clear booster response when the animals were given a second dose of vaccine [20] or infected with BRSV 3 weeks after a single dose of the vaccine [19]. In this study calves were euthanised for post mortem examination at 7 or 10 days post infection, therefore no booster antibody response could be detected but it can be speculated that the animals would have reacted with a booster response.
A very interesting result in this study was the priming of the cellular immune response by a single dose of vaccine in the absence of a measurable antibody response. Kerkhofs and colleagues [20] also reported that the cellular immune response, determined by lymphoproliferation, was induced by the same inactivated BRSV vaccine. When discussed in the literature, the role of CD8 cells in BRSV infection is very controversial. While it has been reported that CD8 responses of animals vaccinated with a different inactivated BRSV vaccine did not protect against immunopathology [28], these immune cells seem to play a key role in the control of infection in calves [29]. This is consistent with the reduction of virus excretion with nasal discharge in vaccinated animals when compared to control animals in the study reported here. This result can be considered a key finding in the study, as a reduction in the excretion of challenge virus i) reflects the replication of the virus indicating that the vaccine is protective and ii) is of practical relevance because reduced virus shedding will also limit the risk of infection for in-contact animals.
Post mortem examinations were performed on two different days (7 or 10 days after infection) as it was expected that hardly any BRSV virus would be detectable more than 1 week after infection, while lung pathology would typically develop around 10 days after infection. In general the results of BRSV re-isolation and lung pathology in this study were in line with these expectations.
BRSV was re-isolated from samples of different organs from the respiratory tract of all control animals and from four out of five vaccinated animals. The overall number of positive samples was lower in the vaccinated group indicating that BRSV replication was reduced after vaccination.
This observation was also reflected by the lung pathology. Macroscopic and histological findings in vaccinated animals were minor and not comparable with the findings in lung sections of BRSV infected unvaccinated animals. From the results obtained it cannot be excluded that other pathogenic agents like Mh were also involved. The vaccine also contains Mh antigen and therefore also protects against this pathogen. In general, more virus could be isolated from the animals euthanised seven days after infection while lung pathology seemed to be more severe in the animals euthanised at 10 days after infection. It has been reported earlier that lung pathology in BRSV infection is the consequence of immuno-pathological events rather that direct damage of the virus to the tissues [29].

Conclusion
From the results described, it can be concluded that, a single vaccination with an inactivated BRSV vaccine was able to break through the maternal immunity and induce partial protection in very young calves. It can be speculated that the level and duration of the protection will improve once a second dose of vaccine is given. Therefore under field conditions the recommended vaccination schedule of two doses must be administered.


extfieldsofstudy: Chemistry Medicine

text:
Simultaneous Observation of Tail and Head Movements of Myosin V during Processive Motion*

Processive stepping of myosin Va (myoV) has been tracked by monitoring either the tail position (center of mass) or the position of one or both heads. Here, we combine these two approaches by attaching a quantum dot to one of the motor domains and a bead to the tail. Using laser trapping and total internal reflection microscopy, the position of one head and the tail are observed simultaneously as myoV moves processively on an actin filament bundle against the resistive load of the laser trap. The head moves one step (73 ± 10 nm) for every two steps of the tail (35 ± 9 nm). One tail step occurs concurrently with quantum dot-labeled head movement, whereas the other occurs with movement of the unlabeled head, consistent with a hand-over-hand model. Load increases the probability of the motor taking a back step. The back step is triggered by the motor taking a shorter forward step (head step, 68 ± 11 nm; tail step, 32 ± 10 nm), likely one actin monomer short of its preferred binding site. During a back step, the motor reverses its hand-over-hand motion, with the leading head detaching and reattaching to one of multiple actin sites behind the trailing head. After a back step, the motor can correct its mistake and step processively forward at resistive loads <0.7 piconewton or stall or detach at higher loads. Back stepping may provide a mechanism to ensure efficient cargo delivery even when myoV encounters obstacles within the actin cytoskeletal meshwork or when other motors are attached to the same cargo.

Myosin Va (myoV), 3 a double-headed processive molecular motor, converts chemical energy derived from ATP hydrolysis into ϳ36-nm steps as it transports intracellular cargo along actin filament tracks (1)(2)(3)(4). Each motor domain binds actin and contains a catalytic site. It is followed by a long ␣-helical neck composed of six calmodulin-binding IQ motifs that serves as a lever arm to amplify small nucleotide-induced changes generated within the motor domain. Following the head is an ␣-helical coiled-coil tail that dimerizes the molecule, and a C-terminal globular tail that binds cargo and which is involved in regulating motor activity (for review, see Ref. 5).
For myoV to take multiple forward steps in a hand-overhand fashion without detaching from actin, the mechanochemical cycles of each head must be out of phase and biased so that the rear or trailing head detaches first from actin and is then propelled forward by the lever arm rotation of the strongly bound leading head. This gating between heads probably results from intramolecular strain that occurs as the leading head attempts to complete its powerstroke and take a forward step but is resisted by the attached trailing head that has yet to complete its enzymatic cycle. This interhead coordination is so robust that under unloaded conditions in vitro, myoV is virtually guaranteed to step forward as evidenced by a low probability (0.3%) of backward steps (6). However, intracellular forces will oppose myoV cargo transport as the motor encounters obstacles within the actin cytoskeletal meshwork or when other motors are attached to the same cargo. Based on our own studies and those of others using single molecule laser trapping, resistive forces increase the probability of myoV taking a back step until the motor stalls (equal probability of a forward or backward step), or steps backward continuously in response to super stall forces (6,7). Without knowing the spatial dynamics of the two heads and their relationship to the center of mass of the molecule (the tail) during such backward motions, one cannot distinguish between the motor slipping back along the actin filament versus the motor reversing its normal stepping pattern.
To define the stepping dynamics of the heads during both forward and backward steps and to understand the structural basis for triggering a back step under resistive load, we have simultaneously monitored the position of one of the two heads and the tail, in a combination laser trap and total internal reflectance fluorescence (TIRF) microscope. By attaching a quantum dot (Qdot) to the N terminus of one head, and a polystyrene bead to the C terminus of the motor, the head and tail positions were determined with nanometer resolution as load was applied to the tail (Fig. 1). Using this approach, backward motions of the motor in response to resistive load were characterized as true reversals of the hand-over-hand stepping mechanism. As myoV normally walks along the 36 nm pseudo-repeat of the actin filament with each head stepping on the 13th actin monomer, back steps are triggered when the leading head lands one actin monomer short of its preferred binding site. Following a back step, the motor will either continue its processive forward stepping or dynamically stall before terminating its run, depending on the resistive load. The ability of myoV to take a back step may allow the motor to continue transporting cargo even when physical challenges are encountered within the cell.

EXPERIMENTAL PROCEDURES
Myosin V Preparation-To enable the simultaneous detection of the myoV head and tail, we used an expressed myoV HMM construct containing an N-terminal biotin tag for attachment of a streptavidin Qdot, and a C-terminal YFP for attachment to an anti-fluorescent protein antibody-coated polystyrene bead (8). In later experiments, we used a heterodimeric myoV HMM that had a biotin tag on only one of its two heads, thus facilitating Qdot labeling of only one head. For this construct, one heavy chain had YFP and FLAG tag at the C terminus and an N-terminal biotin motif, whereas the other heavy chain had YFP at the C terminus and an His 8 motif at the N terminus. The heterodimeric construct was purified by a similar protocol described previously (9). The protein was passed first through a His-SELECT nickel affinity column (Sigma-Aldrich) and then through an anti-FLAG column (Sigma-Aldrich) with the final consisting of the heterodimeric motor.
Actin Bundle Preparation-Chicken skeletal actin was labeled with TRITC-phalloidin as described previously (10). Parallel actin bundles were formed by adding expressed fascin to actin filaments as described in Ref. 11. Briefly, 1 M TRITC-labeled actin filaments were mixed with 1 M fascin in buffer containing 25 mM KCl and 25 mM imidazole (pH 7.4) on ice for 1 h. Actin bundles were then perfused into a flow cell and allowed to bind to a cover glass surface coated with N-ethylmaleimide-modified chicken skeletal muscle myosin for 5 min.
The flow cell was then rinsed with buffer A (25 mM KCl, 1 mM EGTA, 4 mM MgCl 2 , and 25 mM imidazole (pH 7.4)) three times to remove unbound actin.
Simultaneous Detection of MyoV Head and Tail Displacements-Beads coated with an anti-YFP antibody (3E6; Invitrogen) for linkage to the myoV tail provide a well defined attachment strategy compared with nonspecific absorption. Beads were prepared as follows: 2.5 l of 75 pM 1-m polystyrene beads (Polysciences, Inc., Warrington, PA) were mixed with 1 l of 50 mg/ml BSA, 4 l of 0.5 mg/ml anti-fluorescent protein antibody (3E6; Invitrogen), 37.5 l of buffer A and incubated on ice for 1 h. The mixture was briefly spun in a microcentrifuge, the supernatant removed, and the pellet resuspended in 50 l of buffer B (buffer A plus 100 units/ml pyruvate kinase, 0.5 mM phosphoenol pyruvate, an oxygen scavenging system (0.1 mg/ml glucose oxidase, 0.18 mg/ml catalase, and 3 mg/ml glucose), 5 M ATP, and 0.1 mg/ml CaM⌬all, a calcium-insensitive calmodulin mutant (10)). To prepare Qdot-labeled myoV, 0.1 mg/ml myoV (homo-or heterodimeric, containing an N-terminal biotin tag and a C-terminal YFP) was diluted 1/50 in buffer A. 2 l of this solution was mixed with 2 l of 0.2 M streptavidin Qdot (655-nm emission; Invitrogen) for 10 min at room temperature, giving a Qdot:myoV ratio of 53:1. This mixture was then further diluted 50 -60-fold, with a final myoV concentration of 60 -72 pM. To conjugate the Qdot-myoV to the bead, 2 l of 60 pM myoV was mixed with 2 l of 3.75 pM bead (precoated with anti-fluorescent protein antibody and BSA) and allowed to incubate for 10 min at room temperature. Finally, this mixture was diluted with 200 l of buffer B and perfused into the flow cell, which had fascin-actin bundles adhered to the coverslip surface (see above).
To image the myoV displacements associated with the Qdot and bead images, the flow cell was placed on the stage of a combined laser trap:TIRF microscope, based on a Nikon TE2000 inverted microscope equipped with a PlanApo 1.49 N.A. objective. The bead with its attached motor was captured by a custom-built laser trap (6). The bead's brightfield image was projected onto a quadrant photodiode detector, providing bead x and y positions that were filtered to 1 kHz and recorded following analog to digital (A/D) conversion. The trap stiffness was calibrated for each bead using the corner frequency from the bead position power spectrum (12,13). The bead-motor complex was brought into contact with an actin bundle on the coverslip surface that was aligned to the x axis of the laser trap. The actin bundle and the myoV Qdot-labeled head were imaged simultaneously through the objective TIRF using a 488-nm argon ion laser for excitation (Spectra Physics, Santa Clara, CA). The fluorescent images of actin and the Qdot were separated by an image splitter (Optical Insights LLC, Santa Fe, NM) and projected onto an intensified CCD camera (XR-Mega S30; Stanford Photonics, Stanford, CA) and recorded as a TIFF stack using Piper Control software (Agile Automation, Inc., Union City, CA) at 10 -30 frames/s. The effective pixel resolution at 2 ϫ 2 binning was 117 nm/pixel. Because the Qdot image and bead position were recorded by separate computers, it was imperative that the two signals be precisely aligned in time. To accomplish this, an external timing pulse for each captured image frame was provided by the camera manufacturer and A/D converted and recorded by the computer that simultaneously recorded the bead position. This provided timing marks on the bead position trace that could be linked to a specific Qdot image frame.
Data Analysis-The TIFF stack of Qdot images was cropped to a smaller size and then analyzed by a custom-written MATLAB program to find the center of the Qdot intensity profile (i.e. point spread function) to a two-dimensional Gaussian distribution (14). This provided subpixel resolution of 6 nm (15). The bead traces were filtered with a 20-Hz low pass Gaussian filter in Clampfit (Axon Instruments, Inc, Foster City, CA). The Qdot and bead position traces were then analyzed by an objective step-finding algorithm to determine the step size and dwell time of a step (16).

Coordinated Head and Tail Movements of MyoV-
The novel feature of this study is the simultaneous monitoring of the head and tail position of a single myoV, as it processively transports its bead cargo against the resistive load of the laser trap ( Fig. 1). When a captured bead and its attached motor were brought into contact with an actin-fascin bundle, myoV stepped along the actin with the expected ϳ72-nm steps, given that only one of the two motor domains is labeled with a Qdot (15). Steps were detected from the bead signal only after myoV took several steps, suggesting that an effective compliance exists between the motor and the bead. The compliance is due to the bead being held at its center by the laser trap while the motor is attached to the bead surface. Thus, as the motor begins to step, it applies a tangential force to the bead circumference, which will rotate the bead about its center in the trap, as we have shown previously (17). This results in no detectable linear displacement of the bead within the trap for the first few steps of the motor. This creates an effective compliance equivalent to ϳ108 nm at maximum (i.e. three motor steps). Once this compliance has been taken up by the motor at loads of ϳ0.1-0.2 pN, bead steps of ϳ36 nm are detected, and there is a strict correlation between the head and cargo signals. The Qdot-labeled myoV head takes one step for every two steps that the cargo is moved, with one of the cargo steps being coincident with the Qdot-labeled head movement, within the 100-ms time resolution of the fluorescence camera system (Fig. 2a). This observation agrees well with a hand-over-hand mechanism for myoV processivity, with the second tail step originating from movement of the unlabeled head during the dwell period of the Qdot-labeled head. A total of 48 beads were captured, but only 24 beads showed a strict correlation between the bead and Qdot movement. By only analyzing data from these 24 beads, we eliminated the potential for multiple motors/bead so that the resulting 617-bead and 330-Qdot steps were generated by single motors that took seven steps on average during a run.
The step displacements of the head (73 Ϯ 10 nm) and cargo (35 Ϯ 9 nm) were well fit by Gaussian distributions (Fig. 3a and Table 1). The size of each cargo displacement was half that of the head, confirming that the 73-nm step of one head moves the center of mass of the molecule (equivalent to the position of the neck-tail junction), by half that distance. The effect of any compliance is minimal given the good correlation between head and cargo movement both temporally and spatially. The observed step amplitudes agree with literature values obtained using single actin filaments, suggesting that our use of actin-fascin bundles to facilitate interaction of the motor with the track did not affect the inherent stepping pattern of the motor. Furthermore, we did not observe any appreciable motion perpendicular to the direction of travel, either in the bead or Qdot signal (data not shown), indicating that the motor traveled along a single actin filament within the bundle.
Backward Steps Are Reversals of Normal Steps-As the myoV stepped forward against the load of the laser trap, backward steps in both the Qdot and bead signals were observed more frequently (Figs. 2, b-d, and 4a). Although previous studies have described backward steps under resistive loads, the experimental setups could only monitor the center of mass of the motor (6, 7). Backward motor displacements could therefore result from a slip, with the two heads forcibly detached from actin, and then reattached behind their previous position on actin. Alternatively, the motor could effectively walk backward by reversing its normal mode of stepping. With positional data for both the head and tail, we can distinguish between these two possibilities.
If backward motions were a slip, then regardless of which head was labeled with a Qdot, a backward bead motion would always be linked to a backward Qdot motion of the same magnitude (Fig. 5a). For a backward step that is a reversal of a forward step, two scenarios are possible depending on whether the head that steps back is labeled with a Qdot (Fig.  5b). If the Qdot is on the leading head taking the back step, the Qdot and bead both change position coincidentally. This was the case for 45% (30 of 66) of the backward steps (Fig. 2b). If the unlabeled head takes a step back, then the bead and unlabeled head will move, but there will be no change in the position of the Qdot-labeled head, as observed in 55% (36 of 66) of the backward steps (Fig. 2c). The observation of a stationary Qdot position provides strong evidence that myoV is truly taking a back step and not slipping along the actin filament. The fact that approximately half of the backward displacements are characterized this way correlates well with an equal probability of the trailing or leading head being Qdot-labeled.

A Shorter Forward Step under Load Triggers a Back
Step-Because the probability of a back step increases with resistive load, the forward step before a back step should provide insight into what triggers the back step. Interestingly, the forward step taken by a myoV head or tail prior to a back step (Qdot, 68 Ϯ 11 nm; bead, 32 Ϯ 10 nm) was significantly shorter (5 nm and 3 nm, respectively; p Ͻ 0.005) than a nor-FIGURE 3. Analysis of various head and tail step sizes. a, Gaussian fits of head and tail normal forward steps and the smaller forward step taken before the back step. Normal steps are shown in red, and the shorter steps before the back step are in blue. The Gaussian fits to the various head and tail steps were all normalized to 100%, with the peak of the fit set to 100 and with the total number of steps, and the number of steps at the highest recorded data point for each condition as follows: normal head steps (299, 109), normal tail steps (598, 136), head steps before a back step (30, 10), tail steps before a back step (52, 11). All fits had r 2 values Ͼ0.96. b, Gaussian fits of tail back steps (r 2 ϭ 0.87) and forward recovery steps (r 2 ϭ 0.92). c, Gaussian fits of head back steps (r 2 ϭ 0.55) and forward recovery steps (r 2 ϭ 0.77). The black dashed line is the fit of one broad Gaussian peak to the head back steps. The green dashed line is the fit of one Gaussian peak to the head recovery step. The Gaussian fit, although poor, is only meant to illustrate that the data are considerably broader than the head steps shown in a.

TABLE 1 Head and tail step sizes
Head position was determined from Qdot signal with tail position determined from bead signal. Mean Ϯ S.D. are from Gaussian fits to the histograms in Fig. 3.

Signal
Step size a

Normal step
Step before a back step Back step Step size is in nm.

Simultaneous Observation of Myosin V Tail and Head Motion
DECEMBER 31, 2010 • VOLUME 285 • NUMBER 53 mal forward step during processive motion (Fig. 3a, Tables 1 and 2). A back step thus appears to be triggered by the leading head landing one actin monomer short of its preferred binding site that results in a full 73-nm step. As a result, the leading head detaches from actin and lands behind the trailing head. The histogram of back step sizes taken by the head is very broadly distributed about a mean of 49 Ϯ 27 nm (Fig. 3c,  dashed line), suggesting that the head can bind to multiple positions along the actin filament. The histogram of the back step sizes measured at the center of mass (bead) is only slightly broader (29 Ϯ 14 nm) than a normal step or the step prior to the back step (Fig. 3b, Tables 1 and 2).

Fate of Motor after a Back
Step Is Load-dependent-Of 71 back steps, 5 resulted in the motor detaching from actin (Fig.  2d), whereas the remaining 66 were followed by a forward step. The step size for this subsequent forward step (recovery step) was 61 Ϯ 22 nm as measured from the Qdot and 31 Ϯ 10 nm as measured from the bead ( Table 1). The fate of the motor following the 71 back steps fell into two categories. In 32 instances the motor continued processive forward stepping (at least two forward steps following the back step). In the remaining 39 instances the motor either stalled by taking alternating backward and forward steps, or detached from actin prior to or after taking its forward recovery step. Because the probability of a back step occurring increases with resistive load (Fig. 4a), we asked whether the fate of the motor following a back step was also load-dependent. The frequency of the motor continuing to processively step, versus stalling or detaching, was plotted as a function of the resistive load that triggered the back step. These relationships were fit by Gaussians with a mean resistive load of 0.7 Ϯ 0.005 pN for continuing to  b, schematic of a back step that is a reversal of a forward step. For a back step, one should observe either 36-72 nm or 36-0 nm pairs for the tail and head, respectively, depending on whether the head that is stepping back is labeled with a Qdot or not.

Signal
Pairs of steps compared Difference p a nm Head Normal step Step before a back step 5.0 0.005 Tail Normal step Step before a back step 2.8 0.00056 Head Step before a back step Back step 19.5 0.0006 Tail Step before a back step Back step 3.3 b 0.0016 a The p values are the results of Student's t test between the pairs. b The difference between this pair of steps was also compared by a pairwise value, which is the fit to the histogram of the pairwise difference between steps. The value was 4.3 Ϯ 5.1 nm.
step, in contrast to a higher mean load of 0.9 Ϯ 0.03 pN when the motor subsequently stalled or detached (Fig. 4b).

Force Dependence of the Dwell Time for Both Forward and Backward
Steps-Because twice as many steps were observed from the bead signal, we analyzed the bead steps to determine the effect of resistive load on the dwell time for both forward (excluding steps prior to and after a backstep) and backward steps (Fig. 4c). These data were fitted to a modified Arrhenius/Eyring formula of the form, in which 0 is the dwell time under no external load, F is the resistive load, and ␦ is the distance over which the load acts. The fit to the forward steps yielded a 0 of 430 Ϯ 76 ms and ␦ of 3.3 Ϯ 0.5 nm, while the fit to the back steps gave a 0 of 220 Ϯ 32 ms and ␦ of 4.5 Ϯ 0.5 nm.

DISCUSSION
The movement of both the head and center of mass (neck/ tail junction) of myoV during processive motion was simultaneously observed for the first time. Head motion was monitored by a Qdot attached to only one of the two motor domains, whereas the center of mass was monitored by the position of a bead attached to the tail. This experimental setup allowed us to show directly that the center of mass of the molecule moves forward by half the distance of each of its heads and that the heads take turns stepping, in agreement with the commonly accepted hand-over-hand model for processive movement (15). When resistive loads are experienced, this process is reversed, allowing the motor to maintain contact with the actin filament by stepping backward rather than slipping along the filament.

Missing the Optimum Spot on Actin Increases Likelihood of a Back
Step-Under unloaded conditions, myoV takes at least 25 consecutive forward steps on average along the actin filament before terminating its run (18). Under the resistive load of the laser trap, the number of steps is dramatically reduced to ϳ7 steps under the conditions of this study. The probability of the motor taking a back step and/or terminating its run increased with load (Fig. 4a). For normal forward steps each head takes an ϳ73-nm step onto the 13th actin monomer, matching the ϳ36-nm pseudo-repeat of the actin filament. Under resistive load, the forward step is reduced in length by the distance equal to one actin monomer (Tables 1 and 2), suggesting that the motor steps one monomer short along the same actin strand and in doing so increases the probability of a backward step (Fig. 6a).
Why does a short forward step trigger a backstep only under load? Under unloaded conditions, shorter steps occur within the normal distribution of forward steps (Fig. 3a), but they do not lead to a back step. Under unloaded conditions, myoV carrying a bead along a suspended actin filament will spiral around the filament. This suggests that the motor will step occasionally on the 11th actin monomer because it would not spiral if it stepped faithfully on each 13th monomer (19). Based on a lever arm model of myoV, it was estimated that nearly four times more energy is required for the leading head to bind onto the 11th actin monomer compared with the 13th, which is limited only by thermal energy (i.e. 1.1 kT, where k is the Boltzmann constant, and T is temperature in Kelvin) when the leading head is in the pre-powerstroke state and the trailing head is in the post-powerstroke state (20). When myoV takes a shorter forward step, an off-axis component to the resistive load would be experienced by the two lever arms (Fig. 6a), which in turn may weaken the actomyosin bond of the leading head. This was recently reported by Ishiwata and co-workers (21), who applied off-axis loads to single headed myoV S1 constructs. In a similar study, Rief and co-workers (22) show that as little as 1.7 pN was sufficient to detach a 6IQ myoV S1 construct in the absence of ATP. Our back step data show directly for the first time that the leading head (Qdot signal) detaches due to externally applied resistive loads in the piconewton range and reattaches behind the trailing head to then become the new trailing head.
A Back Step Is a Reversal of Normal Forward Stepping-Because a back step appears to be a reversal of the hand-overhand stepping pattern, we also assume that the strongly bound trailing head that remains bound to actin during the back step undergoes a reversal of its powerstroke. Powerstroke reversals from a 6IQ myoV S1 construct were observed under resistive loads in the 2-5 pN range, which were as-5 nm

Normal step
Step before a back step 13 11 2.9 nm 29 nm FIGURE 6. Schematic diagram summarizing the types of steps myoV takes. a, difference between a normal step (leading head bound to the 13th actin subunit) and the shorter step myoV takes before a back step (leading head bound to the 11th actin subunit). When viewed down the actin axis, the two lever arms are in the same plane for a normal step, whereas for a step before a back step, there is an azimuthal angle of ϳ28 o between the two lever arms. b, schematic of a back step. The lever arms in green show the possible binding positions of the new trailing head. During a back step, the tail position changes by 29 Ϯ 14 nm, whereas that of the heads is 49 Ϯ 27.

Simultaneous Observation of Myosin V Tail and Head Motion
DECEMBER 31, 2010 • VOLUME 285 • NUMBER 53 sumed to be a post-to pre-powerstroke transition with ADP in the active site (23). Given that our studies were performed at low ATP concentrations (5 M), the powerstroke reversal of the trailing head seen here presumably occurs while the head is in the rigor state. This is based on the observed dwell time for back steps of 220 ms under unloaded conditions, which agrees with that estimated from a second order ATPbinding rate (k ATP ) of ϳ 1 M Ϫ1 s Ϫ1 (2) and 5 M ATP. The similarity of the load-dependence of the back step and forward step dwell times seen here, together with additional data under load from Kad et al. (6), suggests that the kinetic state of the motor following a back step is probably rate limited by the same process that limits a forward step. This suggests that a back step returns the motor to the same or a comparable "waiting" state that the motor adopts during normal processive stepping. The back step must therefore involve a reversal of the trailing head's post-powerstroke state to a pre-powerstroke-like conformation, but without ADP in the active site. If the powerstroke is not fully reversed to a normally occupied pre-powerstroke state with ADP and P i in the active site, it may account for the smaller than expected 29-nm back step of the center of mass of the motor compared with the 32-nm forward step that preceded the back step (Table 1). In addition, detachment of the leading head, which is presumably in the prepowerstroke conformation with ADP, or ADP and P i in the active state, is then followed by the head reattaching in the postpowerstroke ADP state behind the trailing head. This is supported by a similar distance parameter for the back step and forward step dwell time (Fig. 4c), both of which are consistent with the distance parameter of 4.3 nm reported for the kinetics of the ADP release step following the powerstroke (24).

Position of Detached Head during and after a Back
Step-During a back step, the leading head detaches and reattaches behind the stationary actin-bound head. Given the broad distribution of back step sizes taken by the Qdot-labeled head, the detached head has the capacity to reattach to one of multiple binding sites behind the stationary head (Fig. 6b). The probability of the head binding to any given actin monomer will be determined by geometric constraints and the energy required to overcome the intramolecular strain needed to bend the lever arms as the motor adopts its new conformation (20).
After the back step, the motor then dwells for a period that is limited by the rate of ATP binding to the new trailing head. At this point the motor can resume forward stepping given that the resistive loads used here are well below the 5-pN superstall loads required to force the motor to step backward continuously (7). The next forward recovery step effectively returns the motor to its position on actin that initially triggered the back step, when considering all of the data (Table  1). After the recovery step the motor either continues stepping processively or stalls and detaches, with the fate of motor determined by the resistive load (Fig. 4b). Presumably, at loads Ͻ1 pN, the forward recovery step returns the head to the preferred 13th actin monomer, thus allowing the motor to resume normal processive stepping. For higher loads, the motor most likely takes a short forward recovery step once again, resulting in a higher probability of another back step (i.e. stall) or run termination.

CONCLUSIONS
Our results suggest that myoV is designed to manage effectively the complexities of the intracellular environment, having the capacity to alter its stepping pattern as necessary when presented with resistive loads. MyoV takes a back step to compensate for a less than optimal forward step, in essence attempting to correct its "mistake." If the motor were to encounter an obstacle such as another motor or an actin-associated protein while transporting cargo, then the motor could effectively back up and try stepping forward again or dissociate if a barrier on the actin filament precludes its ability to continue. Either scenario would help prevent myoV from getting stuck on an actin track when cargo delivery is required.


extfieldsofstudy: Biology Medicine

text:
How Do Cells Make Decisions: Engineering Micro- and Nanoenvironments for Cell Migration

Cell migration contributes to cancer metastasis and involves cell adhesion to the extracellular matrix (ECM), force generation through the cell's cytoskeletal, and finally cell detachment. Both adhesive cues from the ECM and soluble cues from neighbouring cells and tissue trigger intracellular signalling pathways that are essential for cell migration. While the machinery of many signalling pathways is relatively well understood, how hierarchies of different and conflicting signals are established is a new area of cellular cancer research. We examine the recent advances in microfabrication, microfluidics, and nanotechnology that can be utilized to engineer micro- and nanoscaled cellular environments. Controlling both adhesive and soluble cues for migration may allow us to decipher how cells become motile, choose the direction for migration, and how oncogenic transformations influences these decision-making processes.

Introduction
Cell migration is an essential element in development, tissue repair and immune surveillance but can become aberrant in cancer leading to malignant invasion of local tissues and metastasis in distant organs. Cell migration is a complex process that integrates numerous extracellular stimuli but also provides feedback to the microenvironment, which can modulate the structure and chemical signature of the extracellular matrix (ECM). Extracellular stimuli can be chemical cues released from cells of the same or different cell type; adhesive cues in the form of cellular ligands that are part of or attached to the ECM or neighbouring cells as well as mechanical forces and structural features within tissue and the ECM. All these parameters contribute to the initiation of migration from a quiescent state, direction and speed of migration and interactions with bystander cells and ECM components.
One can distinguish between different modes of cell migration with mesenchymal and amoeboid cell migrations being two modes of single cell migration. Mesenchymal cell migration, as seen in fibroblast, is characterized by an elongated morphology and firm adhesion to the microenvironment. Amoeboid cell migration such as that of lymphocytes, has dynamic focal complexes and high degree of deformability [1]. Collective cell migration has a similar mode of migration to mesenchymal cell migration but retains cell-cell contacts with neighbouring cells. Such collective cell migration can be seen in bulk cancer cell migration and migrating epithelial cells during wound healing [1,2]. Switching between collective and single cell migration is an important regulator of cancer metastasis and is often associated with altered gene expression.
There are general similarities in single cell migration across cell types, although different cell types have unique features and characteristics. Here, we briefly summarize the general stages of migration since they are frequently used to describe and classify migratory behaviour in cell studies with engineered environments. Cell migration can be divided into four phases: polarisation, protrusion, traction, and disassembly ( Figure 1).
In response to extracellular cues, the cell polarises where specific proteins and lipids accumulate asymmetrically in the anterior and posterior of the cell. During the polarization phase, proteins, lipids and organelles are rearranged in the opposite poles in the cell in order to perform their specific functions in cell migration. Often recruited by ligandreceptor interaction, markers for the leading edge in mesenchymal migration are phosphoinositide 3-kinases (PI3K) [3], the gangliosides GM1 and GM3 [4], phospholipase D [5] while tensin homolog deleted on chromosome 10 (PTEN) is located at the rear [6]. In amoeboid migration, GM3 is also located at the leading edge but GM1 locates to the rear [4]. Rearrangement of organelles also contributes to persistent polarisation. The microtubule organising centre (MTOC) and the Golgi are typically located on the side of the nucleus that faces the leading part of migrating cells [7]. Most polarised cells have a ruffled, fan-shaped protrusion at the leading edge and traction at the rear. At this stage, the cell establishes firm connection with the surrounding matrix or substratum in the form of a nascent adhesion spot, called focal complex and mature adhesion sites called focal adhesion. Attachment of actin stress fibres to focal adhesions, in conjunction with molecular motors, generates forces that move the cell body forward. Finally, a new adhesion forms at the leading edge and old focal adhesions at the rear are disassembled allowing the cell to advance.

Soluble Cues and Directed Cell Migration
Cell signalling molecules such as growth factors, hormones, chemokines, and microbial epitopes can trigger cell migration. These ligands can activate transmembrane receptors such as receptor tyrosine kinases, cytokine receptors and G protein coupled receptors. Triggering of these cell surface receptors recruits signalling proteins to the activation site and consequently intracellular signalling cascades. If the ligands for these receptors are spatially encoded, these processes initiate cell polarization and define the direction of migration towards higher ligand densities.  Figure 2: Directed migration through microchannels engineered with soft lithography techniques. Cancerous cells (red) show different morphology and migratory behaviour than non-cancerous cells (blue) when cocultured in a microchannels of a particular geometry resulted in directed migration [15]. Soft lithography can be used to engineer the microchannels in rigid or soft materials.
The classic chamber to measure directed cell migration, or chemotaxis, is the transwell Boyden chamber. With the transwell Boyden chamber cells are plated on a porous membrane and the chemoattractant is placed in a chamber beneath the membrane. Hence, cells are attracted to migrate through the porous membrane. Unfortunately, these chambers do not allow the visual inspection of the locomotion and are typically used to quantify the percentage of migrating cells. Various microscope setups [8] for directed cell migration have been designed; for example, micropipettes can be used as a point source for chemoattractants or an agarose gel in the so called "under-agarose" assay, in which cells and chemoattractant in solution, are placed inside wells cast into an agarose layer [9]. In all of these designs, the chemoattractant can freely diffuse in solution, which makes it difficult to accurately delivery a known concentration to the migrating cell.
Recently, microfluidic applications and soft lithography technique have been employed to delivery soluble cues to migrating cells [10,11]. In microfluidics, the fluid flow is controlled by viscous force (laminar flow) and in some cases, cells or proteins placement is controlled by electrical stimulation [12,13]. Microfluidics channels are traditionally made of silicon, glass and other rigid materials but more recently elastomeric polymers such as poly(dimethylsiloxane) (PDMS) have begun to be extensively used. To engineer the microfluidics channels ( Figure 2) microcontact printing, which is part of the suite of soft lithography methods but no longer requires soft stamps, is particularly useful because it transfers patterns by stamping and is therefore a nonphotolithographic technique [14].
Because microfluidic channels can be designed in various sized, this approach is suitable for study of single cell migration [15] and cell motility in bulk [11]. Various types of fluidic and fluid-driven mechanical stimuli can be applied for creating gradients, in the microfluidic channel, of growth factors [16], chemoattractants and chemorepellents [10,17], or drugs [18]. In addition, modifications of microfluidics design can be used to study the effect of channel topography, surface pattern, and surface dimensions on cell migration. For instance, the biased migration response of two different cell types was recently reported using microratchet channels.
Interestingly, cancerous cells showed different morphology during migration than noncancerous cells expanding their protrusion pass the boundary of trapezium opening while non-cancerous fibroblast lamellipodia do not broaden its lamellipodia to the trapezium border but anchor to the nearby spike on the trapezium opening [15] (Figure 2).
Other variables can be controlled using microfluidics devices such as adhesive cues [19], shear stress [20], and oxygen level [21]. Microchannels were recently used to test how migration speed is determined by the deformability of cancer cells [22]. This study also highlighted the different motions of cells in confined 3D environment compared to migration over flat surfaces [22].

Cell Adhesion and Integrins
Focal adhesions are the sites of cell connection to ECM where the cell's actin cytoskeleton is tethered to the ECM's nanofibres. The physical link between the outside and inside of the cell is achieved by transmembrane proteins, mainly integrins. Integrins are members of an α/β heterodimeric receptor family [23] (Figure 3). In mammals, there are 18 identified α subunits and 8β subunits that can combine to give 24 distinct heterodimers. Many integrins are expressed in a low-affinity binding state. However, cells can change integrin conformation and hence affinity in response to cellular stimulation, a process termed integrin activation. The signals leading to integrin activation are referred to as "inside-out signalling". Integrin activation results in renewed probing of the cell's environment and, when activated integrins become engaged to their ligands, focal adhesion formation. Thus, focal adhesions contain a high concentration of activated and engaged integrins. In response to growth factors, many cells alter both the repertoire and affinity of integrins.
In addition to the inside-out signalling, focal adhesions control a range of cell activation responses, such as cell polarization and migration, membrane trafficking, cell cycle progression, gene expression, and oncogenic transformation [24,25]. Focal adhesions are large protein complexes, which initiate "outside-in signalling" involving the phosphorylation of focal adhesion kinase (FAK), p130 Cas, Src and other tyrosine kinases, phosphorylation of the structural membrane protein caveolin-1 (Cav1), phosphoinositide (PI) 3-kinases, the kinases Erk, JNK, and p38 MAP kinases, as well as small GTPases [26,27] (Figure 3). In addition, actin-regulating proteins such as vinculin, paxillin and talin link integrin complexes to actin stress fibres. Focal adhesions are also connected to growth factors signalling. For example, vascular endothelial growth factor (VEGF) stimulation of endothelial cells results in the translocation of its type 2 receptors (VEGFR2, KDR/Flk-1) from caveolae to focal adhesions. Dimerization of the receptors initiates autophosphorylation but it is at FA where VEGF receptors induce signalling cascades [28]. In return, VEFGR2 also activates integrins αvβ3, α5β1, and α2β1 [29], and the Rho GTPase Rac1 bridging signalling activities with focal adhesion organization and actin restructuring [30][31][32].
Curiously, integrins have no intrinsic enzymatic activity yet in many cases, integrins enable growth factor signalsthat is, growth factor signalling does not occur unless integrins are occupied. Continuing with the example of endothelial cells, αvβ3 antagonists can inhibit angiogenesis [33], indicating the importance of these integrins in angiogenesis [34]. However, integrins can also suppress growth factor signalling. Genetic deletion of αv integrins has only modest effects on angiogenesis [35] while genetically deleting integrins β3 and β5 enhances normal and pathological angiogenesis [36]. Transdominant integrin inhibition, a form of integrin crosstalk, may account for some of the effect of αvβ3 ligands and antagonists on angiogenesis. There is now considerable evidence that physical and topographical characteristics of integrin ligands regulate integrin function (reviewed recently in [37]).

Model Substrata to Study Cell Attachment and Migration
Because of the crucial role of focal adhesions in both cell adhesion and signal transduction, extensive research has been undertaken to understand the molecular architecture of adhesion sites. Fluorescence microscopy studies are the methods of choice because they enable insights into intact, live cells.
To identify the molecular architecture of focal adhesions, one needs to create surfaces on which the interactions with integrins can be precisely controlled and which mimic the arrangement of adhesive sites in the ECM. Coating glass or plastic surfaces with ECM proteins was the starting point used to identify integrin specificities but because neither the amount nor the location of the ECM protein can be controlled, molecular mechanisms are difficult to identify with this approach. A characteristic feature of the ECM is the periodic nature of integrin ligands. Native collagen, for example, has a fibril structure with each fibril displaying a periodicity of ∼67 nm [38]. This periodicity can increase to 250 nm fibrous collagen [39]. Similarly to collagen, fibronectin is organized into nanoscale patterns, exhibiting paired fibrils with characteristic spacings of 156, 233, 304, and 373 nm [40] while integrin binding sites with fibronection fibre, the tripeptide arginine-glycine-aspartic acid or RGD, have a periodicity below 70 nm. Hence, it is desirable to engineer surfaces on which integrin ligands have precise intermolecular distances of <5 nm to >500 nm.
Adsorbing matrix proteins and modifying polymers provide insufficient control over ligand presentation while microcontact printing and dip-pen lithography achieve at best patterns of 100 nm separation. The first attempt to control the presentation of RGD ligands at the surface was to covalently graft RGD peptides onto a polymer substrate [41,42]. Using these surfaces it was found that cell-substrate interactions depended on RGD density. However, in this approach, RGD peptides were attached to the heterogeneous environment of the polymer gel. As a consequence, the number of RGD peptides accessible by cells differed from the total of RGD within the gel, making it impossible to precisely determine the effective density. Later, this strategy was refined to probe for the optimal RGD spacing for cell adhesion and spreading using glass substrates modified with a polyethylene oxide (PEO) polymer to which RGD peptides were grafted [43] and star-shaped PEO molecules controlling the number of RGD peptides per macromolecule [44]. These studies revealed RGD spacing of 440 nm was sufficient for fibroblast spreading but focal adhesion formation required higher RGD densities [43] or a clustered ligand arrangement [44]. Microcontact printing [45], combined with selfassembled monolayer chemistry [46], and block copolymer nanolithography [47,48] can space integrin ligands closer than 200 nm apart. When spacing integrin ligands that preciously, one has to ensure that the surface between the ligands is sufficiently passivated so that cells specifically interact with the presented ligands and not with the underlying surface. Oligo(ethylene oxide) been shown to be effective in preventing protein absorption [49] and cell adhesion [50] and gold surfaces modified with poly(ethylene glycol)-terminated alkanethiol monolayers [49] ensure specific interactions between the integrins and RGD ligands. Such surface modification strategies demonstrated the importance of density [43,44,51], affinity [52], and spatial organization [44,53] of RGD ligands on cell adhesion and spreading. However, because gold surfaces quench fluorescence, these studies were limited to morphological descriptions.
Spatz and coworkers used block copolymer nanolithography (BCN) to position gold nanodots with high precision at 28, 58, 73, 85, and 108 nm spacing with each nanodot engaging only a single integrin [54,55]. The analysis of focal adhesion dynamics on homogeneously spaced [47], disordered [56] or gradients [48] of cyclic RGD peptide revealed that RGD-to-RGD spacing that exceed >70 nm result in less cell spreading, higher focal adhesion turnover and more erratic cycles of membrane protrusion and retraction in fibroblasts. This detailed analysis of focal adhesion proteins and turnover suggested that there is an optimal RGD density for integrin engagement. Importantly, the 10-200 nm scale of ligand spacing is physiologically relevant as nanoscaled and periodic spacing of integrin ligands below 100 nm is found in fibronectin and collagen fibers [57,58]. In conclusion, cells have the remarkable ability to sense variation in spacing of integrin ligands on the nanometre scale [37].
In contrast to 2D surfaces, our understanding of the effect of surface topography on cell adhesion and migration is limited although substrate topography could affect the ability of cells to orient and migrate as well as influencing the organization of their cytoskeleton [59]. It was found that on striated substrates, endothelial cells, smooth muscle cells, and fibroblasts align and migrate along grooves and that cell orientation is increased with smaller lateral spacing and increasing depth of the grooves present at the surface [60]. The choice of substrate and the chemical patterns on surfaces [61] may also be important. This issue has mainly been addressed by varying the chemical composition of the substrate itself using different metal alloys [62] or polymers [63] but these substrata were devoid of integrin ligands. In contrast, coating titanium with RGD proved to stimulate bone cell adhesion, but the interfaces lacked control over peptide surface density [64]. Particularly for cancer research, model substrata with controllable flexibility, topography, Journal of Oncology 5 chemical modifications, and nano-scaled patterning would provide a better understanding of the impact of the cell's microenvironment on cell migration.

Conclusion
Recent advances in microfluidics, nanopatterning and surface chemistry have afforded unique insights into how cells sense and respond to their local environment [37]. Microfabrication enabled the engineering of microfluidic channels of various topography and shown that the direction of migration is determined by protrusion morphology [15]. Cell adhesion onto 2-dimensional surfaces revealed that cells adhere and spread differently if the density [47], patterning [48], or degree of ordering [56] of the integrin ligands is altered on the nanometre to micrometer scale [65] influencing the distribution of focal adhesion proteins [47] and structuring of the actin cytoskeleton [66]. Only by integrating these advance to, for example, examine cell migration over previously engineered 2D surface in microfluidics devices, or modifying the surface chemistry of microfluidics channels can we understand how cells integrate soluble and adhesive cues in their decision making processes. The fundamental insights into cell adhesion and migration will add the engineering of appropriate cellular environments in medical implants [65], biosensors [67], and tissue materials [68].