scholarly journals Individual Molecular Motors use Low Forces to bypass Roadblocks during Collective Cargo Transport

2021 ◽  
Author(s):  
Saurabh Shukla ◽  
Alice Troitskaia ◽  
Nikhila Swarna ◽  
Barun Kumar Maity ◽  
Marco Tjioe ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Force Sensor ◽  
The Other ◽  
Cargo Transport ◽  
Different Types ◽  
Multicolor Fluorescence ◽  
High Throughput Assay

AbstractA cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesins in vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expected to be generally useful for many different types of molecular motors.

scholarly journals An automated in vitro motility assay for high-throughput studies of molecular motors

Lab on a Chip ◽  
2018 ◽  
Vol 18 (20) ◽  
pp. 3196-3206 ◽  
Author(s):  
Till Korten ◽  
Elena Tavkin ◽  
Lara Scharrel ◽  
Vandana Singh Kushwaha ◽  
Stefan Diez
Keyword(s):  
High Throughput ◽  
Molecular Motors ◽  
Lab On A Chip ◽  
Force Generation ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Cargo Transport

Molecular motors, essential to force-generation and cargo transport within cells, are invaluable tools for powering nanobiotechnological lab-on-a-chip devices.

scholarly journals Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

2017 ◽  
Author(s):  
César Díaz-Celis ◽  
Viviana I. Risca ◽  
Felipe Hurtado ◽  
Jessica K. Polka ◽  
Scott D. Hansen ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Complex Dynamics ◽  
Critical Concentration ◽  
Polarized Growth ◽  
Gtp Hydrolysis ◽  
Filament Dynamics ◽  
Mixed Polarity ◽  
Self Association

AbstractBacteria of the genusProsthecobacterexpress homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). ThebtubABgenes likely entered theProsthecobacterlineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterizedin vitrobMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm ofProsthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.ImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

scholarly journals A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (16) ◽  
Author(s):  
Julia Alterman ◽  
Andrew Coles ◽  
Lauren Hall ◽  
Neil Aronin ◽  
Anastasia Khvorova ◽  
...  
Keyword(s):  
High Throughput ◽  
Cortical Neurons ◽  
Primary Cortical Neurons ◽  
High Throughput Assay

scholarly journals In VitroAdherence of Oral Bacteria to Different Types of Tongue Piercings

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Lucas Pereira Borges ◽  
Julio Cesar Campos Ferreira-Filho ◽  
Julia Medeiros Martins ◽  
Caroline Vieira Alves ◽  
Bianca Marques Santiago ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Bacterial Adhesion ◽  
Oral Bacteria ◽  
The Other ◽  
Control Group ◽  
Colony Forming Units ◽  
Different Types ◽  
Scanning Electron

The purpose of this work was to verifyin vitroadherence ofE. corrodensandS. oralisto the surface of tongue piercings made of surgical steel, titanium, Bioplast, and Teflon. For this, 160 piercings were used for the count of Colony Forming Units (CFU) and 32 piercings for analysis under scanning electron microscopy. Of these, 96 (24 of each type) were individually incubated in 5 mL of BHI broth and 50 μL of inoculum at 37°C/24 h. The other 96 piercings formed the control group and were individually incubated in 5 mL of BHI broth at 37°C/24 h. Plates were incubated at 37°C/48 h for counting of CFU/mL and data were submitted to statistical analysis (pvalue<0.05). ForE. corrodens, difference among types of material was observed (p<0.001) and titanium and surgical steel showed lower bacterial adherence. The adherence ofS. oralisdiffered among piercings, showing lower colonization (p<0.007) in titanium and surgical steel piercings. The four types of piercings were susceptible to colonization byE. corrodensandS. oralis, and bacterial adhesion was more significant in those made of Bioplast and Teflon. The piercings presented bacterial colonies on their surface, being higher in plastic piercings probably due to their uneven and rough surface.

scholarly journals Intracellular cargo transport by single-headed kinesin motors

2019 ◽  
Vol 116 (13) ◽  
pp. 6152-6161 ◽  
Author(s):  
Kristin I. Schimert ◽  
Breane G. Budaitis ◽  
Dana N. Reinemann ◽  
Matthew J. Lang ◽  
Kristen J. Verhey
Keyword(s):  
Intracellular Transport ◽  
Motor Coordination ◽  
Optical Trap ◽  
Coiled Coil ◽  
Force Output ◽  
Cellular Assays ◽  
Cellular Events ◽  
Cargo Transport ◽  
In Cells

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.

In vitro to in vivo concordance of a high throughput assay of bone marrow toxicity across a diverse set of drug candidates

2009 ◽  
Vol 188 (2) ◽  
pp. 98-103 ◽  
Author(s):  
Andrew J. Olaharski ◽  
Hirdesh Uppal ◽  
Matthew Cooper ◽  
Stefan Platz ◽  
Tanja S. Zabka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Throughput ◽  
Bone Marrow Toxicity ◽  
Drug Candidates ◽  
High Throughput Assay

scholarly journals Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions

2002 ◽  
Vol 12 (3) ◽  
pp. 487-492 ◽  
Author(s):  
N. Doi ◽  
H. Takashima ◽  
M. Kinjo ◽  
K. Sakata ◽  
Y. Kawahashi ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Fluorescence Labeling ◽  
Vitro Analysis ◽  
High Throughput Assay ◽  
In Vitro Analysis

Evaluation of mesoporous silica nanoparticles-based nanoantibiotics and capsaicin on E. coli and S. aureus biofilms

2020 ◽  
Author(s):  
Anna Slita ◽  
Prakirth Govardhanam ◽  
Ida Opstad ◽  
Didem Sen Karaman ◽  
Jessica Rosenholm
Keyword(s):  
Mesoporous Silica ◽  
Silica Nanoparticles ◽  
High Throughput ◽  
Mesoporous Silica Nanoparticles ◽  
Inhibitory Effect ◽  
E Coli ◽  
Resazurin Assay ◽  
Antibiotic Drug ◽  
High Throughput Assay

&lt;p&gt;&lt;strong&gt;Introduction&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;Since antibiotics were discovered, bacteria have demonstrated the ability to develop resistance by many different mechanisms. According to WHO reports from 2014, there has been an alarming increase in the antibiotic resistant bacterial strains in most parts of the world&lt;sup&gt;1&lt;/sup&gt;. Our previous results showed that a nanoantibiotic (NAB) design created in our laboratory&lt;sup&gt;2&lt;/sup&gt;, composed of a cerium oxide core, mesoporous silica shell loaded with capsaicin, and a chitosan coating, are effective against planktonic E. coli. However, most of the pathogenic bacteria form biofilms during infections. That is why the next stage of studying NAB is to determine whether they are effective against biofilms of different species. Moreover, the results of NAB efficiency against planktonic E. coli did not clearly show the contribution of the antibiotic drug component of NAB &amp;#8211; capsaicin. Hence, the first step of the current study is to determine whether and to what degree, mesoporous silica nanoparticles (MSN) &amp;#8211; serving as NAB model in this case - penetrate biofilms as a function of particle shape and surface coating; as well as finding the efficient concentration of capsaicin against E. coli and S. aureus &amp;#160;to optimize the NAB dosing against biofilms.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;&amp;#160;&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;&lt;strong&gt;Aim&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;To check in vitro penetration of MSN on S. aureus biofilm and antibacterial activity of NAB and pure capsaicin on E. coli and S. aureus biofilms.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;&lt;br /&gt;Methods&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;To investigate NAB efficiency on biofilms MBEC-high-throughput assay&lt;sup&gt;3&lt;/sup&gt; was performed. Equal biofilms formed on peg-lids were incubated with different concentrations of NAB and capsaicin. After different time point biofilms were sonicated and plated on agar plated to perform CFU counting. To determine the efficient concentration of capsaicin, biofilms were formed in 12 well plates and then incubated with different concentrations of capsaicin. To visualize inhibitory effect, plating for CFU counting and Resazurin assay were applied. To evaluate the penetration of particles, labeled and non-labeled particles were added to fully grown St. aureus biofilms, incubated and visualized with confocal microscopy and structured illumination microscopy.&lt;/p&gt; &lt;p&gt;&lt;strong&gt;&amp;#160;&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;&lt;strong&gt;Results&lt;/strong&gt;&lt;/p&gt; &lt;ol&gt; &lt;li&gt;Through two different microscopy techniques penetration of particles into biofilm and their localization next to bacteria cells were observed.&lt;/li&gt; &lt;li&gt;In MBEC-high-throughput assay no inhibitory effect of NAB against E. coli biofilms was detected in comparison with untreated bacteria.&lt;/li&gt; &lt;li&gt;Resazurin assay and CFU counting method allowed us to determine the most efficient concentration of capsaicin against E. coli and St. aureus biofilms.&lt;/li&gt; &lt;/ol&gt; &lt;p&gt;&lt;strong&gt;&amp;#160;&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;&lt;/p&gt; &lt;ol&gt; &lt;li&gt;Use of MSN and NAB in particular to deliver active antibacterial agents inside the biofilm is justified.&lt;/li&gt; &lt;li&gt;We cannot claim that NAB does not demonstrate any activity against E. coli biofilms, though we can suggest that the peg-lid set up is not sufficient for the NAB design. Further experiments are required.&lt;/li&gt; &lt;li&gt;The next step is to test different concentrations of NAB against biofilms with more appropriate methods than MBEC-high-throughput assay. These results will allow us to make conclusions about the benefits of NAB in comparison with pure capsaicin.&lt;/li&gt; &lt;/ol&gt; &lt;p&gt;&lt;strong&gt;&amp;#160;&lt;/strong&gt;&lt;/p&gt; &lt;p&gt;&lt;strong&gt;References&lt;/strong&gt;&lt;/p&gt; &lt;ol&gt; &lt;li&gt;Govardhanam, N.P. (2017). Development of nanoantibiotics and evaluation via in vitro and in vivo imaging. University of Turku, Finland.&lt;/li&gt; &lt;li&gt;Ventola, C. Lee.&amp;#160;Pharmacy and Therapeutics&amp;#160;40.4: 277, 2015&lt;/li&gt; &lt;li&gt;Harrison, J. et al., BMC microbiology 5(1), 53, 2005.&lt;/li&gt; &lt;/ol&gt;

scholarly journals High-Throughput Screen for Inhibitors of Transglycosylase and/or Transpeptidase Activities of Escherichia coli Penicillin Binding Protein 1b

2004 ◽  
Vol 48 (1) ◽  
pp. 30-40 ◽  
Author(s):  
B. Chandrakala ◽  
Radha K. Shandil ◽  
Upasana Mehra ◽  
Sudha Ravishankar ◽  
Parvinder Kaur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Antibacterial Agents ◽  
Binding Protein ◽  
Type A ◽  
The Other ◽  
Penicillin Binding Protein ◽  
E Coli ◽  
Comparison Of The Results ◽  
High Throughput Assay

ABSTRACT Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC50s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other β-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC50s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

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