Kinesin-1 motility traced by an activity-based precipitating dye

AbstractKinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412.v1 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

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GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

The Journal of General Physiology ◽

10.1085/jgp.200910314 ◽

2009 ◽

Vol 134 (6) ◽

pp. 489-521 ◽

Cited By ~ 28

Author(s):

Fraser J. Moss ◽

P.I. Imoukhuede ◽

Kimberly Scott ◽

Jia Hu ◽

Joanna L. Jankowsky ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Fluorescent Proteins ◽

Resonance Energy ◽

High Order ◽

Live Cells ◽

Gaba Uptake ◽

Cell Periphery ◽

Wild Type ◽

Gaba Transporter ◽

Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

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Microtubules orchestrate local translation to enable cardiac growth

Nature Communications ◽

10.1038/s41467-021-21685-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Emily A. Scarborough ◽

Keita Uchida ◽

Maria Vogel ◽

Noa Erlitzki ◽

Meghana Iyer ◽

...

Keyword(s):

Protein Synthesis ◽

Cell Periphery ◽

Local Translation ◽

Microtubule Network ◽

Critical Determinant ◽

Translation Rate ◽

Cardiac Growth ◽

Translational Machinery ◽

Microtubule Motor ◽

Discrete Domains

AbstractHypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation – and not simply translation rate – is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.

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slowly growing natural populations. Various approaches have been adopted in order to improve the sensitivity. These have included the use of multiple probes labelled with a single fluor (Lee et al. 1993); or labelled with multiple fluors (Trebesius et al. 1994) and enzyme-linked probes or detection systems that allow signal amplification (Lebaron et al. 1997, Schonhuber et al. 1999). The latter indirect approach not only has the potential for signal amplification, but may also be used in natural samples showing high levels of autofluorescence. Any thorough identification method has to include positive and negative controls. False-positive results may either be caused by cells emitting autofluorescence upon excitation or by nonspecific binding of the probe to nontarget cells. Samples should therefore be checked for autofluorescence before hybridization and a negative control with a fluorescent oligonucleotide not complementary to rRNA has to be applied to check for sequence-independent nonspecific binding. Such non-specific binding may be due to interaction of the dye compound of the probe with hydrophobic cell components. Failures to detect cells containing target sequences (false-negatives) may originate from cells with either low cellular ribosome content or limited permeability of the cell periphery for the fluorescent probe (Manz et al. 1992). With the rapidly expanding database of 16S rRNA sequences, the problem of probe specificity has become more apparent and the design of probes is becoming increasingly difficult. These problems are also applicable to PCR and other oligonucleotide-dependent techniques. The problem of probe specificity may be overcome by using multiple specific oligonucleotide probes targeting different sites on the rRNA molecule and labelled with different fluorochromes. While a single oligonucleotide target sequence may be found in a number of related taxa, the probability that target sites for three designed oligonucleotides are found in a nontarget organism is, however, much reduced.

Recent Advances in Marine Biotechnology, Vol. 8 ◽

10.1201/9781482279986-25 ◽

2003 ◽

pp. 232-232

Keyword(s):

Signal Amplification ◽

Specific Binding ◽

Natural Populations ◽

Negative Control ◽

Target Sequence ◽

Cell Periphery ◽

Nonspecific Binding ◽

Detection Systems ◽

Cell Components ◽

Negative Controls

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Fluorescent Probes with Unnatural Amino Acids to Monitor Proteasome Activity in Real-Time

10.26434/chemrxiv.12043101 ◽

2020 ◽

Author(s):

Breanna L. Zerfas ◽

Rachel A. Coleman ◽

Andres Salazar Chaparro ◽

Nathaniel J. Macatangay ◽

Darci Trader

Keyword(s):

Amino Acids ◽

Small Molecule ◽

Fluorescent Probes ◽

Unnatural Amino Acids ◽

Cell Types ◽

Proteasome Activity ◽

Live Cells ◽

Fluorescent Substrate ◽

The Individual ◽

Small Molecule Modulators

<div> <div> <div> <p>The proteasome is an essential protein complex that, when dysregulated, can result in various diseases in eukaryotic cells. As such, understanding the enzymatic activity of the proteasome and what can alter it is crucial to elucidating its roles in these diseases. This can be done effectively by using activity-based fluorescent substrate probes, of which there are many commercially available that target the individual protease-like subunits in the 20S CP of the proteasome. Unfortunately, these probes have not displayed appropriate characteristics for their use in live cell-based assays. In the work presented here, we have developed a set of probes which have shown improved fluorescence properties and selectivity towards the proteasome compared to other cellular proteases. By including unnatural amino acids, we have found probes which can be utilized in various applications, including monitoring the effects of small molecule stimulators of the proteasome in live cells and comparing the relative proteasome activity across different cancer cell types. In future studies, we expect the fluorescent probes presented here will serve as tools to support the discovery and characterization of small molecule modulators of proteasome activity. </p> </div> </div> </div>

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Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6052-6063.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6052-6063

Author(s):

R Kapeller ◽

R Chakrabarti ◽

L Cantley ◽

F Fay ◽

S Corvera

Keyword(s):

Growth Factor ◽

Tyrosine Kinases ◽

Growth Factor Receptor ◽

Endocytic Pathway ◽

Platelet Derived Growth Factor ◽

Cell Periphery ◽

Perinuclear Region ◽

High Concentration ◽

Microtubule Cytoskeleton ◽

Microtubule Network

Phosphatidylinositol (PI)-3' kinase catalyzes the formation of PI 3,4-diphosphate and PI 3,4,5-triphosphate in response to stimulation of cells by platelet-derived growth factor (PDGF). Here we report that tyrosine-phosphorylated PDGF receptors, the p85 subunit of PI-3' kinase (p85), and activated PI-3' kinase are found in isolated clathrin-coated vesicles within 2 min of exposure of cells to PDGF, indicating that both receptor and activated PI-3' kinase enter the endocytic pathway. Immunofluorescence analysis of p85 in serum-starved cells revealed a punctate/reticular staining pattern, concentrated in the perinuclear region and displaying high focal concentration at the centrosome. In addition, partial coalignment of p85 with microtubules was observed after optical sectioning microscopy and image reconstruction. The association of p85 with the microtubule network was further evidenced by the microtubule-depolymerizing drug nocodazole, which caused a redistribution of p85 from the perinuclear region to the cell periphery. Interestingly, the most significant effect of PDGF on the distribution of p85 was an increase in the staining intensity of this protein in the perinuclear region, and this effect was eliminated by prior treatment of cells with nocodazole. These results suggest that PDGF receptor-p85 complexes internalize and transit in association with the microtubule cytoskeleton. In addition, the high concentration of p85 in intracellular structures in the absence of PDGF stimulation suggests additional roles for this protein independent of its association with receptor tyrosine kinases.

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P0511ENHANCER AND SUPER-ENHANCER DYNAMICS IN REPAIR AFTER ISCHEMIC ACUTE KIDNEY INJURY

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa143.p0511 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Julia Wilflingseder ◽

Michaela Willi ◽

Hye Kyung Lee ◽

Hannes Olauson ◽

Jakub Jankowsky ◽

...

Keyword(s):

Acute Kidney Injury ◽

Transcription Factors ◽

Small Molecule ◽

Ischemia Reperfusion Injury ◽

Specific Binding ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Kidney Cortex ◽

Super Enhancer

Abstract Background and Aims The endogenous repair process of the mammalian kidney allows rapid recovery after acute kidney injury (AKI) through robust proliferation of tubular epithelial cells. There is currently limited understanding of which transcriptional regulators activate these repair programs and how transcriptional dysregulation leads to maladaptive repair. Here we investigate the existence of enhancer dynamics in the regenerating mouse kidney. Method RNA-seq and ChIP-seq (H3K27ac, H3K4m3, BRD4, POL2 and selected transcription factors) were performed on samples from repairing kidney cortex 2 days after ischemia/reperfusion injury (IRI) to identify activated genes, transcription factors, enhancer and super-enhancers associated with kidney repair. Further we investigated the role of super-enhancer activation in kidney repair through pharmacological BET inhibition using the small molecule JQ1 in vitro and in acute kidney injury models in vivo. Results Response to kidney injury leads to genome-wide alteration in enhancer repertoire in-vivo. We identified 16,781 enhancer sites (H3K27ac and BRD4 positive, H3K4me3 negative binding) active in SHAM and IRI samples; 6,512 lost and 9,774 gained after IRI. The lost and gained enhancer sites can be annotated to 62% and 63% of down- and up-regulated transcripts at day 2 after kidney injury, respectively. Super-enhancer analysis revealed 164 lost and 216 gained super-enhancer sites at IRI day 2. 385 super-enhancers maintain activity before and after injury. ChIP-seq profiles of selected transcription factors based on motif analysis show specific binding at corresponding enhancer sites. We observed lost enhancer binding of HNF4A and GR mainly at kidney related enhancer elements. In contrast, STAT3 showed increased binding at injury induces enhancer elements. No dynamic was observed for STAT5. Both transcription factor groups show corresponding mRNA changes after injury. Pharmacological inhibition of enhancer and super-enhancer activity by BRD4 inhibition (JQ1: 50mg/kg/day) before IRI leads to suppression of 40% of injury-induced transcripts associated with cell cycle regulation and significantly increased mortality between days 2 and 3 after AKI. Conclusion This is the first demonstration of enhancer and super-enhancer function in the repairing kidney. In addition, our data call attention to potential caveats for use of small molecule inhibitors of BET proteins that are currently being tested in clinical trials in cancer patients who are at risk for AKI. Our analyses of enhancer dynamics after kidney injury in vivo have the potential to identify new targets for therapeutic intervention.

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