The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&amp;M Engineering Experiment Station for funds.

1. תא הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדול תרבויות לילה של המתח הרצוי נושאת את Flic דביק אלל 15,22 ב Tryptone מרק (TB, 1% Peptone, 0.5% NaCl) ואחריו חיסון ב 1: 100 דילול ב 10 TB מ&quot;ל טריים. לגדול התרבות ב 33 מעלות צלזיוס חממה שייקר עד OD 600 = 0.5. </li><li style=";text-align:right;direction:rtl"> גלולה התאים ב 1500 XG במשך 5 - 7 דקות מחדש לפזר את גלולה במרץ ב 10 מ&quot;ל של חיץ תנועתיות מעוקר מסנן (MB; חיץ פוספט 10 מ&quot;מ: 0.05-0.06 M NaCl, 10 -4 M EDTA, 1 מיקרומטר מתיונין, pH 7.0). </li><li style=";text-align:right;direction:rtl"> חזור על שלב 1.2 פעמים נוספות מחדש לפזר את הגלולה הסופית 1 MB המ&quot;ל. </li><li style=";text-align:right;direction:rtl"> גזירה ההשעיה על ידי עובר הלוך ושוב ~ 75 פעמים בין שני מזרקים עם 21 עד 23 מתאמי מד מחובר על ידי צינורות פוליאתילן (7 - 12 ס&quot;מ אורך, 0.58 מ&quot;מ פנימי קוטר). הגבל את הזמן הכולל עבור הגז כדי 30 - 45 s. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה תאים הטעונים ב 1500 XG במשך 5-7 דקות מחדש לפיזורגלולה ב 100 - 500 μL של MB. </li></ol> 2. שקופיות כנות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכין תא הדמיה על ידי הרבדה שנייה דבק דו צדדי בין תלוש-כיסוי שקופיות מיקרוסקופ. עבור מבחני chemotaxis, להעסיק כל תא microfluidic המאפשר חילופי MB וממריצים כימיים. </li><li style=";text-align:right;direction:rtl"> להוסיף 0.01% הפתרון פולי- L- ליזין בתא ואחרי 5 דקות בעדינות ולשטוף את המשטחים עם MB (80 - 100 μL). </li><li style=";text-align:right;direction:rtl"> הוסף 40 μL של ההשעיה תא לתא ולאפשר מספיק זמן עבור התקשרות אל משטח זכוכית (7 - 8 דקות). לזרום החוצה תאים לקלף ידי הוספת 100 μL MB בצד אחד של החדר, תוך פתילת הפתרון עם נייר סינון מהצד השני. </li><li style=";text-align:right;direction:rtl"> להוסיף 10 - 15 μL של חרוזי לטקס לתוך התא ולאפשר החרוזים זמן לפרוע באמצעותם ולצרף אל התאים (7 - 8 דקות). לשטוף בעדינות עם 100 μL של MB, כמתואר בשלב 2.3, להסיר לקלף חרוזים. להשתמש במגוון של חרוז-סיzes עבור הניסויים כל עוד ניגוד טוב זמין. </li></ol> 3. מעקב חרוז <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניח את המדגם על במה מיקרוסקופ ולסרוק את השטח עבור חרוזים מצורפים מנועים. השתמש מטרת שלב 40X לבצע תצפיות למרות מיקרוסקופיה שלב לא הכרחית. לחלופין, להעסיק הדמית שדה בהיר כל עוד ניגוד ראוי נשמר להבחין חרוז בהיר בבירור על רקע כהה. </li><li style=";text-align:right;direction:rtl"> פעם חרוז נבחרת, להזיז את הבמה רוחבית כדי למקם את החרוז בפינה שנקבעה מראש כפי שמוצג באיור 1 ב. חרוזים עמדו באותו בפינה כדי להבטיח כי כיוון הסיבוב של החרוז ידוע כראוי. מסלולו החרוז האידיאלי הוא כ עגול אבל מסלולי אליפטי הם קבילים. </li><li style=";text-align:right;direction:rtl"> שמירה על תדר דגימה גבוה יותר מפי שניים את תדירות הסיבוב של המנוע, כדי למנוע טעויות הקשורות aliasing. בעבודה זו, להשתמש במנוע שהיה מסתובב50 הרץ ו מדגם בתדרים שהיו היה גבוה פי 10 (500 הרץ), כדי לקבל אות חלקה. </li></ol> ניתוח 4. נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מרכז ולהתאים את מתחי מוצא PMT ונכון סְגַלגַלוּת ב המסלולים עם טרנספורמציות המאוחד במידת צורך 23. השתמש ניתוח כוח ספקטרום לקבוע שיעורי סיבוב 17. </li><li style=";text-align:right;direction:rtl"> לקבוע זוויות קוטב, θ (t) = atan (y (t) / x (t)). קבע את הווריאציות במהירויות מנוע ומיתוג לאורך זמן על ידי חישוב ω <img alt="משוואה 1" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14. </li><li style=";text-align:right;direction:rtl"> להעסיק מסנן חציון להחליק את הנתונים מהירי מנוע. חלון מסנן מעל שני סיבובים מלאים מומלץ 23,24. </li></ol>

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The authors have nothing to disclose.

על מנת להקל על מעקב חרוז קשור ואמידה נכונה של מגבילי מנוע, את המידע הבא צריך להיבדק. בעת ביצוע מדידות אלה עם תאים והולקה בשוט, הגז הוא שלב קריטי. גזיזה מפחיתה את נימת flagellar כדי בדל גרידא, ובכך להבטיח כי העומס הצמיג על המנוע הוא בעיקר בשל החרוז ניתן לאמוד בתוך 10% שגיאה 16...

מנועי flagellar לאפשר תאים לשחות ידי החלפת חוטים תאיים סליל. כמות מומנט המנוע יכול לייצר עבור באורך נתון של השוטון (כלומר, את העומס הצמיג) מקובעת את מהירויות השחייה. מצד השני, היכולת שלה כדי להעביר את כיוון הסיבוב שולטת נדידת תאים בתגובת כימיקלים, תהליך המכונה chemotaxis. Chemotaxis ו ארסיות להיות תנועתיות גורמי 1-3, מנועים flagellar מתאפיינים היטב במשך השנים 4. הראיות המצטברות החברה עולה כי המנוע משמש mechanosensor - זה מכנית מזהה נוכחות של 5,6 מצעים מוצקים. יכולת זו מסייעת סביר המעוררים קולוניזציה משטח וזיהומים 5,7. כתוצאה מכך, המנגנונים לפיה מנוע חושי משטחים ויוזם איתות הם בעלי חשיבות 8,9. מנוע flagellar ניתן ללמוד בקלות על ידי קושר את flagellאממ לרוטצית תא מצע והתבוננות. קשירה כזו הושגה לראשונה על ידי סילברמן וסיימון, שעבד עם מוטציה polyhook ב E. coli ו ווים לחבר בהצלחה מצעי זכוכית עם נוגדנים נגד וו 10. את assay הקשורים התאים אפשר לחוקרים ללמוד את התגובות של מתג המנוע למגוון של גירויים כימיים. לדוגמה, סגל ועמיתים לעבודה מגורה תאים קשור כימית בעזרת טפטפות iontophoretic. השינויים המתאימים הטיה CW (את החלק היחסי של מנועים הזמן לסובב עם כיוון השעון, CW) אפשרה להם למדוד את קינטיקה של הסתגלות ברשת chemotaxis 11,12. בעוד assay התא הקשור היה יעיל בחן את תשובות מתג, זה היה מסוגל רק להציע תובנה מכניקת מנוע פני טווח מוגבל של עומסים צמיגים 13. כדי להתגבר על בעיה זו, ריו ועמיתים לעבודה קשורה כדורי, חרוזי גומי כדי נימה השוברים על תאים תקועים על משטחים. החרוזים היואז מעקב באמצעות אינטרפרומטריה גב מוקד עם מלכודות אופטיות חלשות 14. הודות לשיתוף פעולה עם חרוזים בגדלים שונים, חוקרים יכלו ללמוד את המנוע על פני טווח רחב יותר של עומסים. assay זה היה שיפור מאוחר יותר על ידי יואן וברג, אשר פיתח טכניקה חרוז מעקב מבוססי מכפיל בשילוב עם תאורה כהה שדה לייזר. המעקב מופעל שיטתם של nanobeads זהב הקשור כי הם כה זעירים (~ 60 ננומטר) כי ההתנגדויות הצמיגות החיצוניות היו נמוכות לעומת ההתנגדויות הצמיגות הפנימיות סיבוב 15,16. זה הוביל את המידות של מהירויות המרביות השגה ב E. coli (~ 300 הרץ). ב V. alginolyticus, מבחני חרוז דומים מופעלים מדידות של שיעורי ספינינג בעומסים צמיגים ביניים (~ 700 הרץ) 17. בכך שהיא מאפשרת מדידות של תגובות המנוע על פני הטווח האפשרי השלם של המון צמיגה (מאפס עומס לקרוב-דוכן), החרוז-המבחנים ספקו כלי biophysical חשוב להבין את torque דור תהליך 18,19. לאחרונה, שינינו את assay יואן-ברג לכלול פינצטה אופטית שאפשרה לנו ליישם גירויים מכאניים מדויקים מנועים בודדים 6. באמצעות טכניקה זו, הראינו כי-גנרטורים כוח לסובב את המנוע הם mechanosensors דינמי - יעשו שינויים בתגובה לשינויים המון צמיגה. יתכן כי עומס חישה כגון מפעילת התמיינות תאים לתוך חיידקים שורצים, למרות המנגנונים עדיין אינם ברורים. כמו כן סביר להניח כי מנועי flagellar במינים אחרים הם גם mechanosensitive 20, למרות ראיות ישירות חסרות. כאן, אנו דנים מבוססי מכפיל (PMT) הגישה למעקב אחר סיבוב של חרוזים לטקס הרתומים חוטים flagellar 15. בהשוואת מעקב עם מצלמות מהירות, את ההתקנה המכפילה יש יתרון משום שהוא הוא פשוט יחסית כדי לעקוב אחר חרוזים יחידים בזמן אמת ושוב הדורה ארוכהמשא. זה שימושי במיוחד כאשר לומדים ותיק שיפוץ מתחמי מנוע flagellar בשל גירויים סביבתיים 21. למרות שאנחנו פרוטוקולים בפירוט במיוחד עבור E. coli, הם יכולים להתאים בקלות ללימוד מנועים flagellar במינים אחרים.

<table><tbody><tr><td>Poly-L-lysine Solution (0.1%)</td><td>Sigma-Aldrich</td><td>P8920</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&amp;amp;region=US</td></tr><tr><td>Polybead Microspheres</td><td>Polysciences, Inc.</td><td>7307</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&amp;amp;region=US</td></tr><tr><td>1 mL Luer Slip Tip Syringe</td><td>Exel Int.</td><td>26048</td><td>http://www.exelint.com/tuberculin_syringes.php</td></tr><tr><td>Clay Adams Intramedic Luer-Stub Adapter 23-gauge</td><td>Becton, Dickinson and Company</td><td>427565</td><td>http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp</td></tr><tr><td>Polyethylene tubing</td><td>Harvard Apparatus</td><td>59-8325</td><td>http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html</td></tr><tr><td>Photomultiplier Tubes</td><td>Hamamatsu</td><td>R7400U-20</td><td>Spectral response range of 300 to 920 nm, Peak wavelength 630 nm,&amp;nbsp; 0.78 ns response time&amp;nbsp;&lt;br/&gt; http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html</td></tr><tr><td>3 x 1 mm precision slits</td><td>Edmund Optics</td><td>NT39-908</td><td>2 slits mounted at right angles to one another on photomultiplier tubes</td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>TBS 1032B</td><td>Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts&amp;nbsp;&lt;br/&gt; http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope</td></tr><tr><td>8 Pole LP/HP Filter</td><td>Krohn-Hite</td><td>3384</td><td>Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended.&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;br/&gt; http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf</td></tr><tr><td>Optiphot microscope</td><td>Nikon</td><td>NA</td><td>Any upright or inverted phase microscope can be used.&lt;br/&gt; https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754</td></tr><tr><td>50:50 (R:T) Cube Beamsplitter</td><td>ThorLabs</td><td>BS013</td><td></td></tr></tbody></table>

biophysical characterization of flagellar motor functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

ההתקנה המכפילה מוצגת באיור 1 א. חשוב כי יש PMTs רגישויות גבוהות על פני טווח של אורכי גל מפוזרים על ידי החרוזים של עניין. PMTs מועסק כאן לפעול בטווחים הגלויים קרוב אינפרא אדומים, הצליח לזהות אור מפוזר על ידי חרוזים המוארים על ידי מקור אור ההלוגן. ת...

Watch this Scientific Journal Video about Biophysical Characterization of Flagellar Motor Functions at JoVE.com

Biophysical Characterization of Flagellar Motor Functions

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

אפיון Biophysical של פונקציות flagellar Motor

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

biophysical-characterization-of-flagellar-motor-functions

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.2K Views.  Texas A&M University. Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

Video: Biophysical Characterization of Flagellar Motor Functions

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

ייצור Dynein וקינסין הרכבים מוטוריים ב-DNA אוריגמי ננו מבנים עבור תצפית מולקולה אחת

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Biochemistry

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

התאמות ספציפיות מיוסין של במבחנה פלואורסצנטית מיקרוסקופיה מבוססות ישבן

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

תנועתיות של מולקולות בודדות ואשכולות של קינסין-5 Cin8 דו-כיווני מטוהרים מתאי S. cerevisiae

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

ניתוח &quot;מטען מיפוי&quot; המאפיין את ההרכב מולקולרי מוטורס על העברת axonal מטענים שימוש

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

Neuroscience

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

אנליזה של מולקולה בודדת של מנועי משפחת קינסין-3 מטוהרים מסוג Sf9

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

חקר מכניקת אנסמבל מיוזין בצרורות נימה של אקטין באמצעות פינצטה אופטית

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Bioengineering

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

יצירת מוטציות ריקות כדי להבהיר את תפקידם של גליקוזילטרנספראזות חיידקיות בתנועתיות חיידקית

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

Immunology and Infection

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

חקירת תנועתיות מונחית פלאגלה באשריצ'יה קולי על ידי יישום שלוש טכניקות מבוססות בסדרה

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

ניתוח תכונות מכנואנזימטיות של מיוסינים תהליכיים באמצעות ספקטרוסקופיית מהדק כוח מהירה במיוחד

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

Visualizing Bacterial Motility Based on a Color Reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

המחשת תנועתיות חיידקים בהתבסס על תגובת צבע

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...