Supported by grants from the Canadian Institutes of Health Research, the Canadian Stroke Network, and the Alberta Heritage Foundation for Medical Research.

Rotorod walking apparatus The rotorod was a rotating cylinder, 4 cm in diameter, fixed 35 cm above the ground and enclosed by transparent Plexiglas. The rotorod cylinder was covered in textured rubber coating, which facilitates traction. The floor of the apparatus was covered with a layer of foam to prevent injuries when animals fell. A small electric motor provided power to turn the rotorod via a rubber belt. The rotorod was set to rotate once every 5 s. Video recordings and kinematics A high-speed digital video camera was used to film the animals, using a shutter speed of a 1000 of a second. The front, lateral, and rear views were filmed while the animal was walking on the rotorod. A two-arm Nikon, MII cold light source and one set of fiber optic lights were used to provide adequate lighting during recording. The tapes were analyzed frame-by-frame using a Sony DSR 20 DV CAM deck. Video frames were captured using a frame grabber and Macintosh computer. Testing procedure and analysis In order to evaluate the differences between the walking movements of the 6-OHDA and control rats, the video records from post-surgical days 15 - 30 (after any recovery from the lesion might be expected to be largely complete) were examined by two independent observers blind to the experimental conditions. The nigrostriatal 6-OHDA lesion was induced in the right hemisphere, resulting in lasting impairments in the left fore- and hind limbs. A rating scale was developed to rate the rats&rsquo; posture and the stepping movements of the forelimbs (Whishaw et al., 2003). Five components of stepping were rated: <ol> <li>posture, the rating of posture was made by examining the position of the rat on the drum;</li> <li>lift and release, the portion of the stepping cycle in which the paw was lifted from the drum;</li> <li>carry, the portion of the stepping cycle in which the limb was held above the drum as the rat moved forward;</li> <li>advance, the portion of the reach in which the limb was extended to regain contact with the drum; and</li> <li>placement, in which the paw was placed back on the drum.</li> </ol> The following criteria were used to define a normal movement: <ul> <li> Posture: The body of the rat was oriented over the drum with the head held up.</li> <li>Lift and release: The shoulder, elbow and wrist are flexed raising the paw from the surface of the drum.</li> <li>Carry: The paw is held above the surface of the drum, the digits are loosely flexed, and the digit tips are centered on the midline of the body. Advance: The limb is extended forward with the digits moving from a semiflexed position to an extended and open position.</li> <li>Placement: The limb is extended at the shoulder, elbow, and wrist and the digits are opened to place the paw forward and on the surface of the drum, with digit 2 (second digit from the midline) located at approximately the center of the rat&rsquo;s body. </li> </ul> Each movement was rated on a 3-point scale: 1=/the movement resembled that of a control rat, 0.5=/the movement appeared slightly abnormal, and 0=/the movement was clearly abnormal (Whishaw et al., 2003). This rating scale was then used to evaluate five stepping movements from each rat on postoperative days 1, 3, 5, 7, 9, and 15. The scores from the five measures were averaged for each rat on each day, and the results subjected to an ANOVA with days as a repeated measure. Overview of rotorod walking in 6-OHDA and control rats The movement rating scale was applied to the postoperative test sessions for qualitative movement analysis. During the test sessions, the 6-OHDA lesion group received significantly lower scores than did the control group, F(1,8)=33.2, p&lt;0.001, but there was no significant effect of Test Day, nor was there a significant Group by Test Day interaction. The scores of stepping were then separately analyzed with the forelimb as a variable (for the 6-OHDA rats the forelimb contralateral to the lesion versus the forelimb ipsilateral to the lesion; for the control rats, the left paw vs. the right paw). There was a significant effect of Forelimb, F(1,40)=15.8, p&lt;0.01, and there was a significant interaction of Group by Forelimb, F(1,40)=15.0, p&lt;0.01. Follow-up t-tests indicated that the contralateral-to-lesion forelimb of the 6-OHDA group received significantly lower scores than the ipsilateral-to-lesion forelimb or the forelimbs of the control rats, which did not differ. In order to determine which components of the movement contributed to the poor scores obtained by the 6-OHDA group, the measures of posture and the individual components of the stepping cycle from the ipsilateral and contralateral forelimbs of the 6-OHDA group were compared to the values obtained from the control rats. The 6-OHDA group differed in posture and all of the stepping components made by the contralateral limb, but none of the components of the ipsilateral limb (p&lt;0.05). &nbsp; Qualitative and descriptive differences in posture in control and 6-OHDA rats In a control rat, the head is held in a horizontal position, and the supporting forelimb and hind limbs straddle the drum forming a supporting arch. The 6-OHDA rats typically walked with the head down with their impaired forelimbs (facing the viewer) positioned closer together and higher up on the drum surface than occurred for the ipsilateral limbs or the limbs of control rats (see video for a control rat with score "1" for posture, and a 6-OHDA lesion animal with a score of "0"). Typically, the abnormality in posture can be seen at a glance from the relative position of a rat&rsquo;s head. Forelimb stepping In the control sequence, the stepping forelimb is lifted by flexing the shoulder, elbow and wrist. As the limb is lifted, the digits are loosely flexed, and the limb is lifted well above the surface of the drum. When the limb is advanced, it is extended at the shoulders, elbow and wrist and as it is placed the digits are extended and open. In comparison to the control rats, the amplitude of all components of the DA-depleted rats contralateral to lesion limb is attenuated. The limb releases the drum later, is lifted less, and is advanced a shorter distance than is the control forelimb. In addition, when the limb is lifted, the digits are flexed more, and when the limb is placed, the digits are extended and opened less than those of a control rat. The control forelimb is lifted once it reaches the top of the drum, the wrist is flexed to lift the paw up and forward, and the wrist is extended as the limb advances. By contrast, the 6-OHDA rat&rsquo;s contralateral to lesion forelimb is carried further caudally by the movement of the drum, and thus releases later with the wrist still extended. Flexion of the wrist appears to be passive and occurs as the digits are carried backward by the movement of the drum, and when the forelimb is brought forward, the paw and forearm are not lifted clearly off the drum surface. The forelimb of a control rat extends well forward and the digits open as the limb advances. As the limb is placed, it is placed with an arpeggio movement, with digit 5 contacting the surface of the drum first, followed successively by digits 4, 3, and 2. By contrast, the limb of the 6-OHDA rat is lifted and extended less, the digits are extended less, and they contact the surface of the drum almost concurrently with a &lsquo;slap&rsquo;. The video shows forelimb stepping movements in a control animal that were scored as "1", and forelimb stepping movements in a 6-OHDA lesion animal that were mainly scored as "0". Hind limb stepping There were also impairments in the contralateral-to-lesion hind limbs of the 6-OHDA rats. As the control hind limb moves backward, it appears to push against the turning drum and as it does so, the toes rotate outward. As the limb moves forward, the foot is swung outward. The hind limb of the 6-OHDA rat moves directly backward with drum rotation and if anything, turns slightly inward. As the limb moves forward, the foot is carried directly forward. The foot of the control rat swings outward and as it is advanced, the toes are held higher than the ankle, and the toes extend. During the control rat&rsquo;s placement, toe 5 contacts the surface of the drum first and weight is distributed across the toes and palm. In contrast, the toes of the 6-OHDA rat are flexed during carry and placement is made without an arpeggio movement. The toes of the control rat are released in a reversed arpeggio pattern as toe 5 is lifted first and toe 1 is lifted last. This movement was missing in the 6-OHDA lesion rat while the ankle maintained its flexed posture during the carry movement. Again the advance movements of the 6-OHDA rat are attenuated, with the limb less exorotated, lifted less, and advanced less than that of the control rat. As control rats place their toes, they are extended and opened, such that toe 5 points laterally relative to the body of the rat. Toe 5 contacts the drum first followed by the remaining toes such that the rat supported its weight on the toe tips. For the 6-OHDA rat, the toes are somewhat flexed as the foot is carried forward, the toes are lower to horizontal relative to the ankle, there is less opening of the toes when the foot is placed on the drum. Stride length is also somewhat shorter than that of the control rats. In order to quantify the differences between control and 6-OHDA hind limb movements, the components of lift and release, carry, advancement, and placement were rated on a 3-point scale, as were the forelimbs. The measures were made from video records made on post surgical day 20, and three step cycles were rated for each rat. There were significant Group differences, F(1,8)=47.6, p&lt;0.001. Follow-up t-tests on the components indicated that all components of the control rats received significantly higher scores than did the components of the 6-OHDA rats. The video shows hind limb stepping movements in a control animal that were scored as "1". Hind limb stepping movements in the 6-OHDA shown in the video were mainly rated with a score of "0".

<ol>
	<li>Chang, J. W., Wachtel, S. R., Young, D., Kang, U. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+anatomical+characterization+of+forepaw+adjusting+steps+in+rat+models+of+Parkinson's+disease:+studies+on+medial+forebrain+bundle+and+striatal+lesions.">Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson's disease: studies on medial forebrain bundle and striatal lesions.</a> Neuroscience. 88, 617-628 (1999).</li><li>Metz, G. A., Tse, A., Ballermann, M., Smith, L. K., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unilateral+6-OHDA+rat+model+of+Parkinson's+disease+revisited:+an+electromyographic+and+behavioural+analysis.">The unilateral 6-OHDA rat model of Parkinson's disease revisited: an electromyographic and behavioural analysis.</a> Eur J Neurosci. 22, 735-744 (2005).</li><li>Miklyaeva, E. I., Castaneda, E., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+deficits+in+unilateral+dopamine-depleted+rats:+impairments+in+movement+and+posture+and+compensatory+adjustments.">Skilled reaching deficits in unilateral dopamine-depleted rats: impairments in movement and posture and compensatory adjustments.</a> J Neurosci. 14, 7148-7158 (1994).</li><li>Miklyaeva, E. I., Martens, D. J., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+and+compensatory+adjustments+in+spontaneous+movement+after+unilateral+dopamine+depletion+in+rats.">Impairments and compensatory adjustments in spontaneous movement after unilateral dopamine depletion in rats.</a> Brain Res. 68, 23-40 (1995).</li><li>Morrissey, T. K., Pellis, S. M., Pellis, V. C., Teitelbaum, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seemingly+paradoxical+jumping+in+cataleptic+haloperidol-treated+rats+is+triggered+by+postural+instability.">Seemingly paradoxical jumping in cataleptic haloperidol-treated rats is triggered by postural instability.</a> Behav Brain Res. 35, 195-207 (1989).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+locomotor+recovery+following+corticospinal+tract+lesions:+measurement+of+ground+reaction+forces+during+overground+locomotion+in+rats.">Complete locomotor recovery following corticospinal tract lesions: measurement of ground reaction forces during overground locomotion in rats.</a> Behav Brain Res. 103, 45-53 (1999).</li><li>Muir, G. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground+reaction+forces+in+locomoting+hemiparkinsonian+rats:+a+definitive+test+for+impairments+and+compensations.">Ground reaction forces in locomoting hemiparkinsonian rats: a definitive test for impairments and compensations.</a> Exp Brain Res. 126, 307-314 (1999).</li><li>M, O. l. s. s. o. n., Nikkhah, G., Bentlage, C., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forelimb+akinesia+in+the+rat+Parkinson+model:+differential+effects+of+dopamine+agonists+and+nigral+transplants+as+assessed+by+a+new+stepping+test.">Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test.</a> J Neurosci. 15, 3863-3875 (1995).</li><li>Schallert, T., DeRyck, M., Whishaw, I. Q., Ramirez, V. D., Teitelbaum, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+bracing+reactions+and+their+control+by+atropine+and+LDOPA+in+an+animal+analog+of+Parkinsonism.">Excessive bracing reactions and their control by atropine and LDOPA in an animal analog of Parkinsonism.</a> Exp Neurol. 64, 33-43 (1979).</li><li>Schallert, T., Norton, D., Jones, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinically+relevant+unilateral+rat+model+of+Parkinsonian+akinesia.">A clinically relevant unilateral rat model of Parkinsonian akinesia.</a> J Neural Transpl Plasticity. 3, 332-333 (1992).</li><li>Whishaw, I. Q., Gorny, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arpeggio+and+fractionated+digit+movements+used+in+prehension+by+rats.">Arpeggio and fractionated digit movements used in prehension by rats.</a> Behav Brain Res. 60, 15-24 (1994).</li><li>Whishaw, I. Q., O'Connor, W. T., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contributions+of+motor+cortex,+nigrostriatal+dopamine+and+caudate-putamen+to+skilled+forelimb+use+in+the+rat.">The contributions of motor cortex, nigrostriatal dopamine and caudate-putamen to skilled forelimb use in the rat.</a> Brain. 109, 805-843 (1986).</li><li>Whishaw, I. Q., Pellis, S. M., Gorny, B., Kolb, B., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximal+and+distal+impairments+in+rat+forelimb+use+in+reaching+follow+unilateral+pyramidal+tract+lesions.">Proximal and distal impairments in rat forelimb use in reaching follow unilateral pyramidal tract lesions.</a> Behav Brain Res. 56, 59-76 (1993).</li><li>Whishaw, I. Q., Woodward, N. C., Miklyaeva, E., Pellis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+limb+use+by+control+rats+and+unilateral+DA-depleted+rats+in+the+Montoya+staircase+test:+movements,+impairments+and+compensatory+strategies.">Analysis of limb use by control rats and unilateral DA-depleted rats in the Montoya staircase test: movements, impairments and compensatory strategies.</a> Behav Brain Res. 89, 167-177 (1997).</li></ol>

The purpose of the present study was to examine whether the rotorod test for rodent locomotion skill can be used for the qualitative examination of posture, forelimb stepping and hind limb stepping in nervous system injured rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicat...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

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Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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12.9K Views.  University of Lethbridge. The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Video: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

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A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...