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Schematic illustration of the assembly process of casein and kinesin. Kinesin are bound to the surface through interaction with the first adsorbed layer of casein. The second more loosely bound layer of casein interacts with the head domain of the kinesin to promote interaction with microtubules.
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Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functional...
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![Figure 1. Primary structure of the casein from milk; a) αs1-Cs [34], b)...](profile/C-Y-Rosero-Galindo/publication/271768900/figure/fig2/AS:392116573360128@1470499284140/Primary-structure-of-the-casein-from-milk-a-as1-Cs-34-b-aS2-Cs-38-c-b-Cs-39-y_Q320.jpg)
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Citations
... In such cases, the surface may be used as is in conjunction with bovine serum albumin (BSA) 45 or casein 46 to hydrophilize the PDMS surface and prevent nonspecific absorption. In addition, surfaces passivated by BSA or casein can be a base for attaching other proteins 47,48 or further stack functional layers. 49 The protein or linkers will transfer the substrate's strain directly to the sample, allowing strain application to nanoscale samples via large-scale deformations while keeping the surface flat. ...
High-speed atomic force microscopy (HS-AFM) is a powerful tool for studying the dynamics of biomolecules in vitro because of its high temporal and spatial resolution. However, multi-functionalization, such as combination with complementary measurement methods, environment control, and large-scale mechanical manipulation of samples, is still a complex endeavor due to the inherent design and the compact sample scanning stage. Emerging tip-scan HS-AFM overcame this design hindrance and opened a door for additional functionalities. In this study, we designed a motor-driven stretching device to manipulate elastic substrates for HS-AFM imaging of biomolecules under controllable mechanical stimulation. To demonstrate the applicability of the substrate stretching device, we observed a microtubule buckling by straining the substrate and actin filaments linked by α-actinin on a curved surface. In addition, a BAR domain protein BIN1 that senses substrate curvature was observed while dynamically controlling the surface curvature. Our results clearly prove that large-scale mechanical manipulation can be coupled with nanometer-scale imaging to observe biophysical effects otherwise obscured.
... Thirdly, an important aspect of microtubule assays that we have so far ignored is the use of a casein coating on the substrate [66]. The casein layer serves to anchor the kinesin tail and position the kinesin head to better interact with microtubules [67,68]. From the perspective of electrostatic detection, the casein layer displaces a significant thickness (several nm) of the buffer solution in the space between the filament and the nanotube, thereby reducing the screening and increasing the likelihood of detection. ...
... Whole casein is a mixture of four casein sub-groups-α s1 , α s2 , β and κ-in proportions that vary based on species of origin and processing [69]. The composition of the surface-adsorbed casein layer for a microtubule assay can vary and has a significant effect on microtubule binding [67] and motility [70], yet is rarely precisely specified/known for many assays in the literature (e.g., often non-specific descriptors like 'casein-containing buffer' appear in literature protocols). Of the four caseins, only β-casein is well characterized in terms of the formation of a layer on solid surfaces. ...
... Our view of the literature is that there is general agreement that β-casein forms a bilayer on a hydrophilic surface such as SiO 2 . β-casein is amphiphilic [69] and the bilayer forms as a two-step process: (a) first the hydrophilic domain adsorbs to the SiO 2 surface giving a tightly-packed monolayer presenting a hydrophobic outer surface, then (b) a second loosely-packed monolayer forms as hydrophobic domains adsorb to the monolayer presenting a hydrophilic outer surface for the final bilayer [67,68,71,72]. The disagreement in the literature is more related to the thickness and packing density for the β-casein bilayer. ...
Molecular motor gliding motility assays based on myosin/actin or kinesin/microtubules are of interest for nanotechnology applications ranging from cargo-trafficking in lab-on-a-chip devices to novel biocomputation strategies. Prototype systems are typically monitored by expensive and bulky fluorescence microscopy systems. The development of integrated, direct electric detection of single filaments would strongly benefit applications and scale-up. We present estimates for the viability of such a detector by calculating the electrostatic potential change generated at a carbon nanotube transistor by a motile actin filament or microtubule under realistic gliding assay conditions. We combine this with detection limits based on previous state-of-the-art experiments using carbon nanotube transistors to detect catalysis by a bound lysozyme molecule and melting of a bound short-strand DNA molecule. Our results show that detection should be possible for both actin and microtubules using existing low ionic strength buffers given good device design, e.g., by raising the transistor slightly above the guiding channel floor. We perform studies as a function of buffer ionic strength, height of the transistor above the guiding channel floor, presence/absence of the casein surface passivation layer for microtubule assays and the linear charge density of the actin filaments/microtubules. We show that detection of microtubules is a more likely prospect given their smaller height of travel above the surface, higher negative charge density and the casein passivation, and may possibly be achieved with the nanoscale transistor sitting directly on the guiding channel floor. © 2021 The Author(s). Published by IOP Publishing Ltd on behalf of the Institute of Physics and Deutsche Physikalische Gesellschaft.
... The kinesin is known to bind casein surfaces, with the heads pointing at the solution. [42] Then, 10 µL of the MT solution was injected into the channel and incubated for 5 minutes to anchor MTs to the kinesin heads in the presence of AMP-PNP (non-hydrolyzable ATP analog). Unbound MTs were removed by flushing the chamber with 20 µL of the attachment buffer. ...
Microtubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, a label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with enrichment of ordered protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a mechanism of switching from the disassembly to the assembly phase.
... MTs labelled with TRITC were observed using fluorescence microscopy in an assay where MTs glide across the surface propelled by surface-tethered kinesins, as shown in Fig. 1A. A layer of casein on the surface is used to help preserve kinesin functionality 21 . Using a GFP-kinesin-1 fusion protein adsorbed to a coverglass via an anti-GFP antibody, we observed splitting of MTs longitudinally into two fragments, which we propose are PFBs as shown in Fig. 1B. ...
The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.
... Liu et al. [22] proposed that the carboxylate or amine groups of the caseins exhibited an affinity to gold particles. In previous works [23][24][25], it was demonstrated that casein was able to form a bilayer on hydrophilic surfaces. Caseins behave like copolymers consisting of blocks containing hydrophobic and hydrophilic domains. ...
Polylactic acid (PLA) is considered to be a sustainable alternative to petroleum-based polymers for many applications. Using cellulose fiber to reinforce PLA is of great interest recently due to its complete biodegradability and potential improvement of the mechanical performance. However, the dispersion of hydrophilic cellulose fibers in the hydrophobic polymer matrix is usually poor without using hazardous surfactants. The goal of this study was to develop homogenously dispersed cellulose nanowhisker (CNW) reinforced PLA composites using whole milk casein protein, which is an environmentally compatible dispersant.
In this study, whole milk casein was chosen as a dispersant in the PLA-CNW system because of its potential to interact with the PLA matrix and cellulose. The affinity of casein to PLA was studied by surface plasmon resonance (SPR) imaging. CNWs were functionalized with casein and used as reinforcements to make PLA composites. Fluorescent staining of CNWs in the PLA matrix was implemented as a novel and simple way to analyze the dispersion of the reinforcements. The dispersion of CNWs in PLA was improved when casein was present. The mechanical properties of the composites were studied experimentally. Compared to pure PLA, the PLA composites had higher Young's modulus. Casein (CS) functionalized CNW reinforced PLA (PLA-CS-CNW) at 2 wt% filler content maintained higher strain at break compared to normal CNW reinforced PLA (PLA-CNW). The Young's modulus of PLA-CS-CNW composites was also higher than that of PLA-CNW composites at higher filler content. However, all composites exhibited lower strain at break and tensile strength at high filler content.
The presence of whole milk casein improved the dispersion of CNWs in the PLA matrix. The improved dispersion of CNWs provided higher modulus of the PLA composites at higher reinforcement loading and maintained the strain and stress at break of the composites at relatively low reinforcement loading. The affinity of the dispersant to PLA is important for the ultimate strength and stiffness of the composites.
... Whole casein or the substructures of casein were used onto microtubule motility assays to reserve the kinesin functionality. It was found that the adsorbed casein bilayer improves the activity of kinesin, by one of the tightly bound casein layer anchoring the kinesin, while the second loosely bound layer of casein improves the position of kinesin for interaction with micro- tubules [59]. ...
Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively.
... In the gliding motility assay, motility is sustained by first passivating the glass to prevent kinesin's motor domains from becoming inactive when interacting with untreated glass. Passivation of glass can be done with bovine serum albumin (BSA)91011, bovine casein1213141516, a lot of kinesin [17], or other compositions [18]. Bovine casein is the typical surface blocker used, mainly because it works well at passivation and is inexpensive. ...
... Bovine casein is the typical surface blocker used, mainly because it works well at passivation and is inexpensive. Casein is a globular protein that does not have a known crystal structure [17]. Bovine casein is comprised of four major subgroups: a s1 , a s2 , b, and k. ...
... Ozeki et al. showed that two layers of casein form on the glass surface to help support kinesin for motility [12]. Verma et al. [17] also investigated how kinesin and casein interact depending on which casein constituent from bovine milk was used. In their study, they showed that the number of microtubules that landed on the kinesin surface was affected by the casein passivation. ...
In this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays. Whole casein, comprised of α(s1), α(s2), β, and κ casein, supported motility and averaged speeds of 966±7 nm/s. Alpha casein can be purchased as a combination of α(s1) and α(s2) and supported gliding motility and average speeds of 949±4 nm/s. Beta casein did not support motility very well and averaged speeds of 870±30 nm/s. Kappa casein supported motility very poorly and we were unable to obtain an average speed. Finally, we observed that mixing alpha, beta, and kappa casein with the proportions found in bovine whole casein supported motility and averaged speeds of 966±6 nm/s.
... Passivation of glass can be done with bovine serum albumin (BSA) [9][10][11], bovine casein [12][13][14][15][16], a lot of kinesin [17], or other compositions [18]. Bovine casein is the typical surface blocker used, mainly because it works well at passivation and is inexpensive. ...
... Bovine casein is the typical surface blocker used, mainly because it works well at passivation and is inexpensive. Casein is a globular protein that does not have a known crystal structure [17]. Bovine casein is comprised of four major subgroups: α s1 , α s2 , β, and κ. ...
... Ozeki et al. showed that two layers of casein form on the glass surface to help support kinesin for motility [12]. Verma et al. [17] also investigated how kinesin and casein interact depending on which casein constituent from bovine milk was used. In their study, they showed that the number of microtubules that landed on the kinesin surface was affected by the casein passivation. ...
In this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays. Whole casein, comprised of [alpha]~s1~, [alpha]~s2~, [beta], and [kappa] casein, supported motility and averaged speeds of 966 ± 7 nm/s. Alpha casein can be purchased as a combination of s1 and s2 and supported gliding motility and average speeds of 949 ± 4 nm/s. Beta casein did not support motility very well and averaged speeds of 870 ± 30 nm/s. Kappa casein supported motility very poorly and we were unable to obtain an average speed. Finally, we observed that mixing alpha, beta, and kappa casein with the proportions found in bovine whole casein supported motility and averaged speeds of 966 ± 7 nm/s.
... Passivation of glass can be done with bovine serum albumin (BSA) [9][10][11], bovine casein [12][13][14][15][16], a lot of kinesin [17], or other compositions [18]. Bovine casein is the typical surface blocker used, mainly because it works well at passivation and is inexpensive. ...
... Bovine casein is the typical surface blocker used, mainly because it works well at passivation and is inexpensive. Casein is a globular protein that does not have a known crystal structure [17]. Bovine casein is comprised of four major subgroups: α s1 , α s2 , β, and κ. ...
... Ozeki et al. show that two layers of casein form on the glass surface to help support kinesin for motility [12]. Verma et al. [17] also investigated how kinesin and casein interact depending on which casein constituent from bovine milk was used. In their study, they showed that the number of microtubules that landed on the kinesin surface is affected by the casein passivation. ...
In this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays. Whole casein, comprised of [alpha]~s1~, [alpha]~s2~, [beta], and [kappa] casein, supported motility and averaged speeds of966 ± 7 nm/s. Alpha casein can be purchased as a combination of s1 and s2 and supported gliding motility and average speeds of 949 ± 4 nm/s. Beta casein did not support motility very well and averaged speeds of 870 ± 30 nm/s. Kappa casein supported motility very poorly and we were unable to obtain an average speed. Finally,we observed that mixing alpha, beta, and kappa casein with the proportions found in bovine whole casein supported motility and averaged speeds of 966 ± 7 nm/s.
... When casein was added to the motility solution, odd squiggle patterns were seen in the assay. This observation led us to not include casein in our motility assay despite literature recommendations 1 ...
... Very nice work done by Verma et. al.1 (above) shows that microtubules are supported differently in the gliding motility assay dependent on the type of casein used as a passivator. Building on what they have done (below), we investigated the speed changes microtubules glide at depending on what type of passivation is used. ...
The molecular motor kinesin-1, an ATPase, and the substrate it walks along, microtubules, are vital components of eukaryotic cells. Kinesin converts chemical energy to linear motion as its two motor domains step along microtubules in a process similar to how we walk. Cells create systems of microtubules that direct the motion of kinesin. This directed motion allows kinesin to transport various cargos inside cells.During the stepping process, the kinesin motor domains bind and unbind from their binding sites on the microtubules. Binding and unbinding rates of biomolecules are highly dependent on hydration and exclusion of water from the binding interface. Osmotic stress will likely strongly affect the binding and unbinding rates for kinesin and thus offers a tool to specifically probe those steps. We will report the effects of different osmolytes on microtubule speed and other observables in the gliding motility assay.Kinesin’s kinetic core cycle hydrolyzes ATP with the help of a water molecule. Any modification to the water molecules the kinesin is in will change how ATP hydrolyzes and will ultimately affect how kinesin moves along microtubules. We will report preliminary results showing how kinesin is affected when the solvent it is in is changed from light water to heavy water.When used in a surface assay or in devices, the kinesin and microtubule system is also dependent on substrate passivation. Kinesin motor domains do not transport microtubules in the gliding motility assay if kinesin is added to a glass microscope slide that has not been functionalized. Functionalization of the glass slides and slips is typically performed with bovine milk proteins called caseins. Bovine casein is a globular protein that can be broken up into four constituents: αs1, αs2, β, and κ. Each casein constituent affects how kinesin adheres to the glass and ultimately the speed at which microtubules are observed to glide at. Building on the work of Verma et.al., we have found that each constituent individually produces different outcomes in gliding assays. We will present these findings and discuss implications they have for use of gliding assays to study kinesin and use of kinesin-microtubule system in microdevices.[1] Chaen, S, N Yamamoto, I Shirakawa, and H Sugi. 2001. Effect of deuterium oxide on actomyosin motility in vitro. Biochimica et biophysica acta 1506, no. 3: 218-23.[2] Vivek Verma, William O Hancock, Jeffrey M Catchmark, "The role of casein in supporting the operation of surface bound kinesin," J. Biol. Eng. 2008; 2: 14.
Acknowledgements:
This work was supported by the DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008.CORRECTIONS: At the workshop, Erik Schaffer pointed out to us that some of our speed differences (casein data) were surely due to microscope increasing in temperature. I've edited the poster to correct for this. Thanks, Erik!
