The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

ABSTRACTMicrotubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes in particular, during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors’ residence time at the microtubule end. On the microtubule lattice, loads also exponentially decreased the run length and time. However, for the same load, lattice run times were significantly longer compared to end residence times suggesting the presence of a distinct force-dependent detachment mechanism at the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism that unifies the so-called “bump-off” and “switching” models. Understanding the mechanics of kinesin-8’s microtubule end activity will provide important insights into cell division with implications for cancer research.STATEMENT OF SIGNIFICANCEKinesin-8 motors are important for microtubule length regulation and are over-expressed in different types of cancer. Yet, on the molecular level, it is unclear how these motors depolymerize microtubules. Using high-precision optical tweezers, we measured how single yeast kinesin-8 motors, Kip3, interacted with the microtubule end. Interestingly, we found that single Kip3 motors were still motile at the microtubule end. The force dependence of how long single motors were associated with the microtubule end enabled us to estimate what force motors must exert onto each other to achieve the collective microtubule depolymerization speed of many motors. Our data support a collective force-dependent depolymerization mechanism. A better understanding of Kip3’s microtubule end activity has implications for cell division and associated diseases.

Download Full-text

Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces

eLife ◽

10.7554/elife.36764 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 30

Author(s):

Vladimir A Volkov ◽

Pim J Huis in 't Veld ◽

Marileen Dogterom ◽

Andrea Musacchio

Keyword(s):

Residence Time ◽

Dependent Manner ◽

Microtubule Depolymerization ◽

Microtubule Binding ◽

Ndc80 Complex ◽

Multiple Copies ◽

Biased Diffusion ◽

Spindle Microtubules ◽

Incremental Addition ◽

Do So

Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly.

Download Full-text

Force and Stepwise Movements of Gliding Motility in Human Pathogenic Bacterium Mycoplasma pneumoniae

Frontiers in Microbiology ◽

10.3389/fmicb.2021.747905 ◽

2021 ◽

Vol 12 ◽

Author(s):

Masaki Mizutani ◽

Yuya Sasajima ◽

Makoto Miyata

Keyword(s):

Optical Tweezers ◽

Mycoplasma Pneumoniae ◽

Binding Activity ◽

Gliding Motility ◽

Pathogenic Bacterium ◽

Step Size ◽

Bacterial Surface ◽

Human Pathogenic Bacterium ◽

A Cell ◽

Stall Force

Mycoplasma pneumoniae, a human pathogenic bacterium, binds to sialylated oligosaccharides and glides on host cell surfaces via a unique mechanism. Gliding motility is essential for initiating the infectious process. In the present study, we measured the stall force of an M. pneumoniae cell carrying a bead that was manipulated using optical tweezers on two strains. The stall forces of M129 and FH strains were averaged to be 23.7 and 19.7 pN, respectively, much weaker than those of other bacterial surface motilities. The binding activity and gliding speed of the M129 strain on sialylated oligosaccharides were eight and two times higher than those of the FH strain, respectively, showing that binding activity is not linked to gliding force. Gliding speed decreased when cell binding was reduced by addition of free sialylated oligosaccharides, indicating the existence of a drag force during gliding. We detected stepwise movements, likely caused by a single leg under 0.2-0.3 mM free sialylated oligosaccharides. A step size of 14-19 nm showed that 25-35 propulsion steps per second are required to achieve the usual gliding speed. The step size was reduced to less than half with the load applied using optical tweezers, showing that a 2.5 pN force from a cell is exerted on a leg. The work performed in this step was 16-30% of the free energy of the hydrolysis of ATP molecules, suggesting that this step is linked to the elementary process of M. pneumoniae gliding. We discuss a model to explain the gliding mechanism, based on the information currently available.

Download Full-text

Force and step size of gliding motility in human pathogenic bacterium Mycoplasma pneumoniae

10.1101/2021.05.25.445585 ◽

2021 ◽

Author(s):

Masaki Mizutani ◽

Yuya Sasajima ◽

Makoto Miyata

Keyword(s):

Optical Tweezers ◽

Mycoplasma Pneumoniae ◽

Binding Activity ◽

Gliding Motility ◽

Pathogenic Bacterium ◽

Step Size ◽

Bacterial Surface ◽

Human Pathogenic Bacterium ◽

A Cell ◽

Stall Force

Mycoplasma pneumoniae, a human pathogenic bacterium, binds to sialylated oligosaccharides and glides on host cell surfaces via a unique mechanism. Gliding motility is essential for initiating the infectious process. In the present study, we measured the stall force of an M. pneumoniae cell carrying a bead that was manipulated using optical tweezers on two strains. The stall forces of M129 and FH strains were averaged to be 23.7 and 19.7 pN, respectively, much weaker than those of other bacterial surface motilities. The binding activity and gliding speed on sialylated oligosaccharides of the M129 strain were eight and two times higher than those of the FH strain, respectively, showing that binding activity is not linked to gliding force. Gliding speed decreased when cell binding was reduced by addition of free sialylated oligosaccharides, indicating the existence of a drag force during gliding. We detected stepwise movements under 0.2‒0.3 mM free sialylated oligosaccharides. A step size of 14‒19 nm showed that 25‒35 propulsion steps per second are required to achieve the usual gliding speed. The step size was reduced to less than half with the load applied using optical tweezers, showing that a 2.5 pN force from a cell is exerted on a leg. The work performed in this step was 16‒30% of the free energy of the hydrolysis of ATP molecules, suggesting that this step is linked to the elementary process of M. pneumoniae gliding.

Download Full-text

Two dynamin-like proteins stabilize FtsZ rings duringStreptomycessporulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704612114 ◽

2017 ◽

Vol 114 (30) ◽

pp. E6176-E6183 ◽

Cited By ~ 30

Author(s):

Susan Schlimpert ◽

Sebastian Wasserstrom ◽

Govind Chandra ◽

Maureen J. Bibb ◽

Kim C. Findlay ◽

...

Keyword(s):

Cell Division ◽

Protein Interactions ◽

Time Lapse ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Cellular Processes ◽

Organelle Division ◽

Z Ring ◽

A Cell ◽

Massive Cell

During sporulation, the filamentous bacteriaStreptomycesundergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the twoStreptomycesdynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein–protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

Download Full-text

NDC80 clustering modulates microtubule dynamics under force

10.1101/198291 ◽

2017 ◽

Author(s):

Vladimir A. Volkov ◽

Pim J. Huis in't Veld ◽

Marileen Dogterom ◽

Andrea Musacchio

Keyword(s):

Residence Time ◽

Dependent Manner ◽

Microtubule Depolymerization ◽

Microtubule Binding ◽

Multiple Interfaces ◽

Biological Phenomena ◽

Ndc80 Complex ◽

Multiple Copies ◽

Spindle Microtubules ◽

Incremental Addition

AbstractMultivalency, the presence of multiple interfaces for intermolecular interactions, underlies many biological phenomena, including receptor clustering and cytosolic condensation. One of its ultimate purposes is to increase binding affinity, but systematic analyses of its role in complex biological assemblies have been rare. Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved but poorly characterized feature of kinetochores, the points of attachment of chromosomes to spindle microtubules. To address its significance, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. While modules containing a single NDC80 complex were unable to track depolymerizing microtubules, modules with two or more complexes tracked depolymerizing microtubules and stiffened the connection with microtubules under force. Cargo-conjugated modules of divalent or trivalent NDC80 stalled and rescued microtubule depolymerization in a force-dependent manner. Thus, multivalent microtubule binding through NDC80 clustering is crucial for force-induced modulation of kinetochore-microtubule attachments.

Download Full-text

C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons

Human Molecular Genetics ◽

10.1093/hmg/ddab089 ◽

2021 ◽

Author(s):

Eun Seon Kim ◽

Chang Geon Chung ◽

Jeong Hyang Park ◽

Byung Su Ko ◽

Sung Soon Park ◽

...

Keyword(s):

Retinal Degeneration ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Accumulation ◽

Heterozygous Mutation ◽

Pathological Feature ◽

Dependent Manner ◽

Cellular Processes ◽

Drosophila Model ◽

Post Transcriptional Regulation

Abstract RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS—but not with mutated endogenous NLS—potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/−), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/− is protective. Stau+/− also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

Download Full-text

Arginine Decarboxylase Is Essential for Pneumococcal Stress Responses

Pathogens ◽

10.3390/pathogens10030286 ◽

2021 ◽

Vol 10 (3) ◽

pp. 286

Author(s):

Mary Frances Nakamya ◽

Moses B. Ayoola ◽

Leslie A. Shack ◽

Mirghani Mohamed ◽

Edwin Swiatlo ◽

...

Keyword(s):

Stress Responses ◽

Critical Role ◽

Acid Stress ◽

Arginine Decarboxylase ◽

Arginine Deiminase ◽

Dependent Manner ◽

Cellular Processes ◽

Opportunistic Human Pathogen ◽

Thiol Peroxidase ◽

The Impact

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host–pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

Download Full-text

184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0184 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A198-A198

Author(s):

Tingting Zhong ◽

Xinghua Pang ◽

Zhaoliang Huang ◽

Na Chen ◽

Xiaoping Jin ◽

...

Keyword(s):

T Cells ◽

Mixed Culture ◽

Cell Activity ◽

Cross Reactivity ◽

Binding Activity ◽

Jurkat Cells ◽

List Type ◽

Dependent Manner ◽

Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

Download Full-text

Volatile 1-octanol of tea (Camellia sinensis L.) fuels cell division and indole-3-acetic acid production in phylloplane isolate Pseudomonas sp. NEEL19

Scientific Reports ◽

10.1038/s41598-021-82442-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Poovarasan Neelakandan ◽

Chiu-Chung Young ◽

Asif Hameed ◽

Yu-Ning Wang ◽

Kui-Nuo Chen ◽

...

Keyword(s):

Acetic Acid ◽

Cell Division ◽

Petri Dish ◽

Gas Chromatography Mass Spectrometry ◽

Dependent Manner ◽

Tea Leaves ◽

Acetic Acid Production ◽

Iaa Production ◽

Solvent Tolerant ◽

Indole 3 Acetic Acid

AbstractTea leaves possess numerous volatile organic compounds (VOC) that contribute to tea’s characteristic aroma. Some components of tea VOC were known to exhibit antimicrobial activity; however, their impact on bacteria remains elusive. Here, we showed that the VOC of fresh aqueous tea leaf extract, recovered through hydrodistillation, promoted cell division and tryptophan-dependent indole-3-acetic acid (IAA) production in Pseudomonas sp. NEEL19, a solvent-tolerant isolate of the tea phylloplane. 1-octanol was identified as one of the responsible volatiles stimulating cell division, metabolic change, swimming motility, putative pili/nanowire formation and IAA production, through gas chromatography-mass spectrometry, microscopy and partition petri dish culture analyses. The bacterial metabolic responses including IAA production increased under 1-octanol vapor in a dose-dependent manner, whereas direct-contact in liquid culture failed to elicit such response. Thus, volatile 1-octanol emitting from tea leaves is a potential modulator of cell division, colonization and phytohormone production in NEEL19, possibly influencing the tea aroma.

Download Full-text

The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0500 ◽

2004 ◽

Vol 15 (2) ◽

pp. 481-496 ◽

Cited By ~ 34

Author(s):

Josefa Andrade ◽

Hu Zhao ◽

Brian Titus ◽

Sandra Timm Pearce ◽

Margarida Barroso

Keyword(s):

Endoplasmic Reticulum ◽

Conformational Changes ◽

Membrane Trafficking ◽

Secretory Pathway ◽

Network Formation ◽

Binding Protein ◽

Dependent Manner ◽

Microtubule Depolymerization ◽

Independent Manner ◽

Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

Download Full-text