Formation of helical membrane tubes around microtubules by single-headed kinesin KIF1A

Abstract The kinesin-3 motor KIF1A is in charge of vesicular transport in neuronal axons. Its single-headed form is known to be very inefficient due to the presence of a diffusive state in the mechanochemical cycle. However, recent theoretical studies have suggested that these motors could largely enhance force generation by working in teams. Here we test this prediction by challenging single-headed KIF1A to extract membrane tubes from giant vesicles along microtubule filaments in a minimal in vitro system. Remarkably, not only KIF1A motors are able to extract tubes but they feature a novel phenomenon: tubes are wound around microtubules forming tubular helices. This finding reveals an unforeseen combination of cooperative force generation and self-organized manoeuvreing capability, suggesting that the diffusive state may be a key ingredient for collective motor performance under demanding traffic conditions. Hence, we conclude that KIF1A is a genuinely cooperative motor, possibly explaining its specificity to axonal trafficking.

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Endoplasmic reticulum degradation of a subunit of the asialoglycoprotein receptor in vitro. Vesicular transport from endoplasmic reticulum is unnecessary.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48445-1 ◽

1992 ◽

Vol 267 (1) ◽

pp. 5-8 ◽

Cited By ~ 5

Author(s):

L WikstrɆm ◽

H F Lodish

Keyword(s):

Endoplasmic Reticulum ◽

Vesicular Transport ◽

Asialoglycoprotein Receptor ◽

A Subunit

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In Vitro Reconstitution of Dynamic, ER-like, Nanotubular Networks, and of Small, Tubulo-Vesicular Transport Entities by Interactions of Cytoplasmic Dynein and Spectrin with Liposomes

Biophysical Journal ◽

10.1016/j.bpj.2008.12.2613 ◽

2009 ◽

Vol 96 (3) ◽

pp. 507a ◽

Cited By ~ 2

Author(s):

Virgil Muresan ◽

Zoia Muresan

Keyword(s):

Vesicular Transport ◽

Cytoplasmic Dynein ◽

In Vitro Reconstitution

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Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.823 ◽

1991 ◽

Vol 112 (5) ◽

pp. 823-831 ◽

Cited By ~ 31

Author(s):

Y Goda ◽

S R Pfeffer

Keyword(s):

Early Stage ◽

Gradient Centrifugation ◽

Vesicular Transport ◽

Free System ◽

Transport Reaction ◽

Trans Golgi Network ◽

Transport Vesicles ◽

Golgi Network ◽

Gamma S

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.

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Epithelial Cell Chirality Revealed by Three-Dimensional Spontaneous Rotation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805932115 ◽

2018 ◽

Vol 115 (48) ◽

pp. 12188-12193 ◽

Cited By ~ 15

Author(s):

Amanda S. Chin ◽

Kathryn E. Worley ◽

Poulomi Ray ◽

Gurleen Kaur ◽

Jie Fan ◽

...

Keyword(s):

Environmental Factors ◽

Cell Polarity ◽

Three Dimensional ◽

Intrinsic Property ◽

Cell Aggregates ◽

Mechanistic Studies ◽

Directional Bias ◽

Self Organized ◽

Dependent Cell

Our understanding of the left–right (LR) asymmetry of embryonic development, in particular the contribution of intrinsic handedness of the cell or cell chirality, is limited due to the confounding systematic and environmental factors during morphogenesis and a ack of physiologically relevant in vitro 3D platforms. Here we report an efficient two-layered biomaterial platform for determining the chirality of individual cells, cell aggregates, and self-organized hollow epithelial spheroids. This bioengineered niche provides a uniform defined axis allowing for cells to rotate spontaneously with a directional bias toward either clockwise or counterclockwise directions. Mechanistic studies reveal an actin-dependent, cell-intrinsic property of 3D chirality that can be mediated by actin cross-linking via α-actinin-1. Our findings suggest that the gradient of extracellular matrix is an important biophysicochemical cue influencing cell polarity and chirality. Engineered biomaterial systems can serve as an effective platform for studying developmental asymmetry and screening for environmental factors causing birth defects.

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Geometric Effect for Biological Reactors and Biological Fluids

Bioengineering ◽

10.3390/bioengineering5040110 ◽

2018 ◽

Vol 5 (4) ◽

pp. 110 ◽

Cited By ~ 2

Author(s):

Kazusa Beppu ◽

Ziane Izri ◽

Yusuke Maeda ◽

Ryota Sakamoto

Keyword(s):

Biological Fluids ◽

Biological Systems ◽

Self Organization ◽

Active Matter ◽

Confined Space ◽

Self Organized ◽

Motile Cells ◽

Recent Developments ◽

A Cell

As expressed “God made the bulk; the surface was invented by the devil” by W. Pauli, the surface has remarkable properties because broken symmetry in surface alters the material properties. In biological systems, the smallest functional and structural unit, which has a functional bulk space enclosed by a thin interface, is a cell. Cells contain inner cytosolic soup in which genetic information stored in DNA can be expressed through transcription (TX) and translation (TL). The exploration of cell-sized confinement has been recently investigated by using micron-scale droplets and microfluidic devices. In the first part of this review article, we describe recent developments of cell-free bioreactors where bacterial TX-TL machinery and DNA are encapsulated in these cell-sized compartments. Since synthetic biology and microfluidics meet toward the bottom-up assembly of cell-free bioreactors, the interplay between cellular geometry and TX-TL advances better control of biological structure and dynamics in vitro system. Furthermore, biological systems that show self-organization in confined space are not limited to a single cell, but are also involved in the collective behavior of motile cells, named active matter. In the second part, we describe recent studies where collectively ordered patterns of active matter, from bacterial suspensions to active cytoskeleton, are self-organized. Since geometry and topology are vital concepts to understand the ordered phase of active matter, a microfluidic device with designed compartments allows one to explore geometric principles behind self-organization across the molecular scale to cellular scale. Finally, we discuss the future perspectives of a microfluidic approach to explore the further understanding of biological systems from geometric and topological aspects.

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Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells

PLoS ONE ◽

10.1371/journal.pone.0249686 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249686

Author(s):

Diána Hudecz ◽

Sara Björk Sigurdardóttir ◽

Sarah Christine Christensen ◽

Casper Hempel ◽

Andrew J. Urquhart ◽

...

Keyword(s):

Endothelial Cells ◽

Transferrin Receptor ◽

Vesicular Transport ◽

Brain Diseases ◽

Brain Endothelial Cells ◽

Binding Studies ◽

Wide Range ◽

Murine Brain ◽

The Brain

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

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Isolation, spectroscopic characterization, X-ray, theoretical studies as well as in vitro cytotoxicity of Samarcandin

Bioorganic Chemistry ◽

10.1016/j.bioorg.2016.03.005 ◽

2016 ◽

Vol 66 ◽

pp. 27-32 ◽

Cited By ~ 6

Author(s):

Salar Hafez Ghoran ◽

Vahideh Atabaki ◽

Esmaeil Babaei ◽

Seyed Reza Olfatkhah ◽

Michal Dusek ◽

...

Keyword(s):

Spectroscopic Characterization ◽

In Vitro Cytotoxicity ◽

Theoretical Studies ◽

X Ray

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

Lab on a Chip ◽

10.1039/c8lc00547h ◽

2018 ◽

Vol 18 (20) ◽

pp. 3196-3206 ◽

Cited By ~ 1

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.

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