scholarly journals Inducible manipulation of motor-cargo interaction using engineered kinesin motors

2021 ◽  
Author(s):  
Jessica J. A. Hummel ◽  
Casper C. Hoogenraad
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Chemical Induction ◽  
Systematic Screening ◽  
Px Domain ◽  
Transport Vesicles ◽  
Polarized Trafficking ◽  
A Cell ◽  
Living Neurons ◽  
Motor Interaction

Molecular motors drive long-range intracellular transport of various vesicles and other cargos within a cell. Identifying which kinesin motors interact with which type of transport vesicles has been challenging, especially in complex neuronal cells. Here, we present a highly adaptable toolbox of engineered kinesin motors to control and interrogate the selectivity and regulation of cargo transport with acute chemical induction. Selectivity of cargo-motor interaction can be addressed by systematic screening of a library of kinesin tails and neuronal cargos. Additionally, our toolbox can be used to study kinesin-cargo regulatory mechanisms, and we found that cargo trafficking by KIF16B is regulated by its PX-domain. Furthermore, our toolbox enables acute manipulation of polarized trafficking in living neurons by stirring transport into axons or dendrites. Engineering kinesin motors provides a powerful tool to map the specificity of interactions between kinesin and cargos, manipulate polarized transport and investigate cargo-motor interaction modes.

Specific KIF1A–adaptor interactions control selective cargo recognition

2021 ◽  
Vol 220 (10) ◽  
Author(s):  
Jessica J.A. Hummel ◽  
Casper C. Hoogenraad
Keyword(s):  
Motor Activity ◽  
Family Member ◽  
Posttranslational Modifications ◽  
Molecular Motors ◽  
Synaptic Vesicles ◽  
Intracellular Transport ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Transport Vesicles

Intracellular transport in neurons is driven by molecular motors that carry many different cargos along cytoskeletal tracks in axons and dendrites. Identifying how motors interact with specific types of transport vesicles has been challenging. Here, we use engineered motors and cargo adaptors to systematically investigate the selectivity and regulation of kinesin-3 family member KIF1A–driven transport of dense core vesicles (DCVs), lysosomes, and synaptic vesicles (SVs). We dissect the role of KIF1A domains in motor activity and show that CC1 regulates autoinhibition, CC2 regulates motor dimerization, and CC3 and PH mediate cargo binding. Furthermore, we identify that phosphorylation of KIF1A is critical for binding to vesicles. Cargo specificity is achieved by specific KIF1A adaptors; MADD/Rab3GEP links KIF1A to SVs, and Arf-like GTPase Arl8A mediates interactions with DCVs and lysosomes. We propose a model where motor dimerization, posttranslational modifications, and specific adaptors regulate selective KIF1A cargo trafficking.

scholarly journals Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1391
Author(s):  
Brent Race ◽  
Katie Williams ◽  
Chase Baune ◽  
James F. Striebel ◽  
Clayton W. Winkler ◽  
...  
Keyword(s):  
Prion Disease ◽  
Molecular Motors ◽  
Prion Diseases ◽  
Mouse Strains ◽  
Survival Times ◽  
Knock Out ◽  
Transport Vesicles ◽  
The Central Nervous System ◽  
Knock Out Mice ◽  
Scrapie Strains

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c−/− mice.

Sterol and lipid trafficking in mammalian cells

2006 ◽  
Vol 34 (3) ◽  
pp. 335-339 ◽  
Author(s):  
F.R. Maxfield ◽  
M. Mondal
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Vesicular Transport ◽  
Cholesterol Content ◽  
Cellular Functions ◽  
Lipid Trafficking ◽  
Sterol Transport ◽  
A Cell ◽  
Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

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 Neuronal receptors display cytoskeleton-independent directed motion on the plasma membrane

2018 ◽  
Author(s):  
R. D. Taylor ◽  
M. Heine ◽  
N. J. Emptage ◽  
L. C. Andreae
Keyword(s):  
Plasma Membrane ◽  
Neuronal Activity ◽  
Particle Tracking ◽  
Hippocampal Neurons ◽  
Intracellular Transport ◽  
Transmembrane Proteins ◽  
Single Particle Tracking ◽  
Directed Motion ◽  
Surface Movement ◽  
Transport Vesicles

AbstractDirected transport of transmembrane proteins is generally believed to occur via intracellular transport vesicles. However, using single particle tracking in rat hippocampal neurons with a pH-sensitive quantum dot probe which specifically reports surface movement of receptors, we have identified a subpopulation of neuronal EphB2 receptors that exhibit directed motion between synapses within the plasma membrane itself. This receptor movement occurs independently of the cytoskeleton but is dependent on cholesterol and is regulated by neuronal activity.

scholarly journals Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA

2015 ◽  
Author(s):  
John Salogiannis ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Coiled Coil ◽  
Leading Edge ◽  
Time Lapse ◽  
Early Endosome ◽  
Organelle Transport ◽  
The Novel ◽  
Coiled Coil Region ◽  
Early Endosomes

Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast to this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we find that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport where peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.

Computational analysis of hereditary spastic paraplegia mutations in the kinesin motor domains of KIF1A and KIF5A

2020 ◽  
Vol 19 (06) ◽  
pp. 2041003
Author(s):  
Vidhyanand Mahase ◽  
Adebiyi Sobitan ◽  
Christina Johnson ◽  
Farion Cooper ◽  
Yixin Xie ◽  
...  
Keyword(s):  
Free Energy ◽  
Protein Stability ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Computational Analysis ◽  
Binding Free Energy ◽  
Membrane Vesicles ◽  
Energy Calculation ◽  
Missense Mutations ◽  
Kinesin Motor

Hereditary spastic paraplegias (HSPs) are a genetically heterogeneous collection of neurodegenerative disorders categorized by progressive lower-limb spasticity and frailty. The complex HSP forms are characterized by various neurological features including progressive spastic weakness, urinary sphincter dysfunction, extra pyramidal signs and intellectual disability (ID). The kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motors involved in intracellular transport. Kinesins directionally transport membrane vesicles, protein complexes, and mRNAs along neurites, thus playing important roles in neuronal development and function. Recent genetic studies have identified kinesin mutations in patients with HSPs. In this study, we used the computational approaches to investigate the 40 missense mutations associated with HSP and ID in KIF1A and KIF5A. We performed homology modeling to construct the structures of kinesin–microtubule binding domain and kinesin–tubulin complex. We applied structure-based energy calculation methods to determine the effects of missense mutations on protein stability and protein–protein interaction. The results revealed that the most of disease-causing mutations could change the folding free energy of kinesin motor domain and the binding free energy of kinesin–tubulin complex. We found that E253K associated with ID in KIF1A decrease the protein stability of kinesin motor domains. We showed that the HSP mutations located in kinesin–tubulin complex interface, such as K253N and R280C in KIF5A, can destabilize the kinesin–tubulin complex. The computational analysis provides useful information for understanding the roles of kinesin mutations in the development of ID and HSPs.

Application of Quasi-Steady-State Methods to Nonlinear Models of Intracellular Transport by Molecular Motors

2017 ◽  
Vol 79 (9) ◽  
pp. 1923-1978 ◽  
Author(s):  
Cole Zmurchok ◽  
Tim Small ◽  
Michael J. Ward ◽  
Leah Edelstein-Keshet
Keyword(s):  
Steady State ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Nonlinear Models ◽  
Quasi Steady State

scholarly journals Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

2006 ◽  
Vol 74 (10) ◽  
pp. 5617-5624 ◽  
Author(s):  
Fang Cai ◽  
Carrie B. Adrion ◽  
James E. Keller
Keyword(s):  
Culture Medium ◽  
Reaction Model ◽  
Cultured Neurons ◽  
Total Inhibition ◽  
Botulinum Neurotoxins ◽  
A Cell ◽  
Proteolytic Activities ◽  
Living Neurons ◽  
Subsequent Addition

ABSTRACT Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

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