Journal: The Journal of Biological Chemistry

Article Title: MAP7 regulates organelle transport by recruiting kinesin-1 to microtubules

doi: 10.1074/jbc.RA119.008052

Figure Lengend Snippet: MAP7 targets bidirectional cargoes to the microtubule plus-end by increasing the number of engaged kinesin motors. A, isolated phagosomes containing fluorescent beads were positioned on polarity-marked microtubules, and the forces were measured using an optical trap. These isolated phagosomes are transported by teams of kinesin-1, kinesin-2, and dynein motors (14). B, MAP7 binds in static patches (Fig. S1J; Movie S6), whereas tau forms both static and diffusive patches along the microtubule lattice (Fig. S1K). The mean dwell time of MAP7 is 114.5 ± 6.16 s (n = 221 events from 11 recordings, two independent experiments), compared with 14.9 ± 1.19 s for tau (n = 128 events from 26 recordings, two independent experiments). C, force traces were acquired at 2 kHz and median-filtered at 20 Hz. We observe force events directed toward both the minus-end (i, iii, and vii) and plus-end (ii, v, and vi) and also bidirectional force events (iv and viii) (0 nm: n = 689 events from 38 recordings, 11 independent experiments; 10 nm MAP7: n = 787 events from 40 recordings, 11 independent experiments). D, maximum forces for all trap displacements greater than 300 ms (top) and 500 ms (bottom) in duration were included in the histogram. Consistent with previous results (14, 30), plus-end–directed force events consist of unitary stall forces of kinesin-1 and kinesin-2, events where the motors detach before reaching their stall force, and rare events driven by multiple kinesins. Minus-end–directed forces indicate events driven by teams of several dynein motors (Fig. S1C). The frequency and magnitude of kinesin-driven forces increase in the presence of 10 nm MAP7. In response, dynein-mediated forces are reduced. The Bayesian information criterion was used to determine the optimal number of components to describe the force histograms (Fig. S1F). Mean forces for plus-end directed motors are 1.57, 2.38, and 4.5 and for minus-ended motors are 1.14, 1.6, 2.8, and 5.8 pN. With MAP7, mean forces of the multicomponent fits for plus-end–directed forces are 1.4, 2.4, 4.5, and 7.3 pN, and for minus-end–directed forces, they are 1.4, 2.7, and ≥9 pN (p < 0.001; Kolmogorov–Smirnov test; Fig. S5A). E, teams of kinesin and dynein motors remain engaged to the microtubule under load for longer durations in the presence of MAP7. The color of the trajectory indicates the duration of a stall event. The duration of force events is significantly longer for kinesin-driven events with MAP7 ( = 1.5 s, = 2.5 s, p < 0.01), whereas the duration of dynein-driven forces is slightly decreased ( = 2.26 s, = 1.8 s, p = not significant) (Fig. S5C). F, comparing the duration of events of similar maximum force indicates that MAP7 does not alter the duration of events driven by similar numbers of motors. G, the relative binding rate was calculated as the reciprocal of the dwell time (green periods) preceding a force event (yellow). H, MAP7 increases the relative binding rate of plus-ended motors on MAP7 decorated microtubules by 28% (<π0 nM,+> = 0.89 s−1, <π10 nM,+> = 1.14 s−1; <π0 nM,−> = 1.05 s−1, <π10 nM,−> = 1.15 s−1). Error bars, S.E.

Article Snippet: MAP7 expression and purification MAP7 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BC052637","term_id":"30962906","term_text":"BC052637"}} BC052637 ) was cloned from pEGFP-MAP7 (Addgene plasmid 46076) into the baculovirus expression vector pFastBac with N-terminal FLAG and SNAP tags.

Techniques: Isolation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: MAP7 regulates organelle transport by recruiting kinesin-1 to microtubules

doi: 10.1074/jbc.RA119.008052

Figure Lengend Snippet: MAP7 recruits kinesin-1 to microtubules. A and B, MAP7 recruits full-length kinesin-1 to microtubules in a dose-dependent manner (see also Fig. S2B). MgADP-bound kinesin-1 is in a weakly bound state. Kinesin-1 hydrolyzes MgATP to drive processive movement. C, when present at high levels, MAP7 increases kinesin-1 run lengths by up to ∼30%, possibly due to rapid re-attachment (average run length: 0 nm = 2033 ± 493.8 nm; 5 nm = 1640 ± 463 nm; 10 nm = 1800 ± 937 nm; 25 nm = 2227 ± 411.2 nm; 75 nm = 3002 ± 265.5 nm). D, the frequency of both processive and diffusive motility of kinesin-1 increases to a similar degree with increasing MAP7 concentration (0 nm MAP7: n = 15 events, three recordings; 5 nm MAP7: n = 17 events from three recordings; 10 nm MAP7: n = 21 events from three recordings; 25 nm MAP7: n = 37 events from three recordings; 75 nm MAP7: n = 70 events from three recordings). E and F, beads transported by ∼1–2 kinesin-1 motors were positioned on the microtubule using a weak optical trap (k ∼0.004 pN/nm) and imaged as they moved along the microtubule. G, long, directed events toward the microtubule plus-end indicate motility by multiple kinesin-1 motors. H, teams of kinesin-1 motors are more processive on MAP7-decorated microtubule (I and J). The average run length of kinesin-1 motors with MAP7 increases by ∼8-fold (average run length (0 nm) = 595 ± 60 nm; average run length (10 nm) = 5320 ± 890 nm), whereas the velocity is unaffected (average velocity (0 nm) = 220 ± 16 nm/s; average velocity (10 nm) = 292 ± 45 nm/s) (0 nm: n = 72 events from 21 trajectories, two independent experiments; 10 nm MAP7: n = 32 events from 18 trajectories, two independent experiments). Error bars, S.E.

Article Snippet: MAP7 expression and purification MAP7 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BC052637","term_id":"30962906","term_text":"BC052637"}} BC052637 ) was cloned from pEGFP-MAP7 (Addgene plasmid 46076) into the baculovirus expression vector pFastBac with N-terminal FLAG and SNAP tags.

Techniques: Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: MAP7 regulates organelle transport by recruiting kinesin-1 to microtubules

doi: 10.1074/jbc.RA119.008052

Figure Lengend Snippet: MAP7 increases the forces generated by teams of kinesin-1. We measured forces by single kinesin-1 motors and teams at three motor densities (ratio of motors to beads in the conjugation reaction). A, 3300 kinesin-1s/bead resulted in motility driven by a single kinesin-1, where <50% of beads interacted with the microtubule; G, 6600 kinesin-1s/bead resulted in motility due to 1–2 engaged kinesin-1 motors; M, 13,000 kinesin-1s/bead resulted in 2–3 kinesin-1 motors. B and C, on beads driven by single kinesin-1 motors, force distributions indicate unitary stall force of kinesin-1 and substall detachment forces (mean forces of the multicomponent fits Fcomp = 1.3 and 4.03 pN). With the addition of MAP7, substall detachment events decrease, and the frequency of unitary stall force events due to single kinesin-1 increases (Fcomp = 4.23 and ≥9 pN) (p < 0.001; Kolmogorov–Smirnov test; Fig. S5B) (0 nm: n = 444 events from 25 recordings, four independent experiments; 10 nm MAP7: n = 536 events from 25 recordings, four independent experiments). H and I, on beads driven by ∼1–2 kinesin-1s, force distributions show three distinct populations: forces at unitary stall force of kinesin-1, detachment forces, and rare multimotor events (Fcomp = 1.27, 3.47, and 7 pN). With the addition of MAP7, detachment and low force events decrease, whereas the frequency of unitary stall force events increases (Fcomp = 1.17, 4, and 7 pN) (p < 0.001; Kolmogorov–Smirnov test; Fig. S5B). The frequency and magnitude of multimotor force events remain unchanged (0 nm: n = 479 events from 26 recordings, four independent experiments; 10 nm MAP7: n = 623 events from 24 recordings, four independent experiments). N and O, on beads driven by ∼2–3 kinesin-1 motors, force distribution shows three distinct populations: forces at unitary stall force of kinesin-1, low-force events, and multimotor events (Fcomp = 1.24, 3.64, and 7.5 pN). With MAP7, we observe a shift toward frequent high-force events driven by multiple engaged kinesin-1s (Fcomp = 4.24, 5.97, and 7.29) (p < 0.001; Kolmogorov–Smirnov test; Fig. S5B) (0 nm: n = 485 events from 23 recordings, three independent experiments; 10 nm MAP7: n = 378 events from 22 recordings, three independent experiments). D, J, P, and Fig. S3, the duration of stall events at a given force is not strongly influenced by MAP7, indicating that MAP7 does not affect the processivity of single kinesin motors. Single kinesin-1 motors (E) and teams of 1–2 kinesin-1 motors (K) bind much faster to MAP7-decorated microtubules. Q, the increase in binding rate is not observed for larger teams of 2–3 kinesin-1s, likely because attachment is no longer limited by the single-motor binding rate when many motors are available for binding (single kinesin-1: 0 nm = 0.77 s−1 and 10 nm = 0.95 s−1; teams of ∼1–2 kinesin-1s: 0 nm = 1.04 s−1 and 10 nm = 1.55 s−1; ∼2–3 kinesin-1s: 0 nm = 1.24 s−1 and 10 nm = 1.35 s−1). F, L, R, the force–velocity curves indicate that more kinesin-1 motors are simultaneously engaged when MAP7 is present, as indicated by higher velocities at the same load. Error bars, S.E.

Article Snippet: MAP7 expression and purification MAP7 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BC052637","term_id":"30962906","term_text":"BC052637"}} BC052637 ) was cloned from pEGFP-MAP7 (Addgene plasmid 46076) into the baculovirus expression vector pFastBac with N-terminal FLAG and SNAP tags.

Techniques: Generated, Conjugation Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: MAP7 regulates organelle transport by recruiting kinesin-1 to microtubules

doi: 10.1074/jbc.RA119.008052

Figure Lengend Snippet: MAP7 directs transport toward the microtubule plus-end. A, we extended the mathematical model proposed by Muller et al. (40) to describe the interaction between teams of kinesin-1, kinesin-2, and dynein motors based on single-motor parameters including the motor stall force, detachment force, and unbinding and binding rates (14). We modeled the effect of MAP7 by increasing the binding rate of single kinesin-1 molecules by the same amount as we measured in optical trapping measurements. Kinesin-2 and dynein were assumed to be unaffected by MAP7. B, the increase in kinesin-1 binding rate biases simulated trajectories toward the plus-end (number of simulated trajectories, n = 100). C, we calculated run length as distance between reversals (29) and consider events >400 nm as processive. Plus-end–directed run lengths increase by ∼24%, whereas minus-end–directed run lengths decrease by ∼24%. D, the model results suggest that the shift in phagosome transport toward the microtubule plus-end that we observe can be described by simply increasing the kinesin-1 binding rate with no direct effect on kinesin-2 or dynein. E, the number of engaged kinesin and dynein motors changes in the presence of MAP7. A greater number of plus-ended motors are simultaneously engaged and exerting force, and as a result, minus-ended motors are under higher loads and are less processive. F, MAP7 and tau exhibit distinct localizations in neurons. Whereas MAP7 is enriched at axonal branches (21), tau is localized in a gradient along the axon. Thus, MAP7 might target cargoes to the microtubule plus-end at branch sites, whereas tau directs distal cargoes toward the cell body. Error bars, S.E.

Article Snippet: MAP7 expression and purification MAP7 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BC052637","term_id":"30962906","term_text":"BC052637"}} BC052637 ) was cloned from pEGFP-MAP7 (Addgene plasmid 46076) into the baculovirus expression vector pFastBac with N-terminal FLAG and SNAP tags.

Techniques: Binding Assay

Journal: Biology

Article Title: A Novel Vitronectin Peptide Facilitates Differentiation of Oligodendrocytes from Human Pluripotent Stem Cells (Synthetic ECM for Oligodendrocyte Differentiation)

doi: 10.3390/biology10121254

Figure Lengend Snippet: Induction and expansion of hPSC-derived OPCs. ( A ) A bright-field image of OPCs at day 12 of differentiation. Scale bar: 20 µm. (Inset) A representative image of NESTIN-positive cells in the culture on day 12. ( B , C ) Immunofluorescent images of OLIG2, A2B5 and NG2. ( D ) Quantification of cells positive for OLIG2, A2B5, and NG2. ( E ) Quantitative gene expression of OPC markers ( OLIG2 and PDGFRα ) and a pluripotency marker ( NANOG ). ( F , G ) Representative images of OLIG2, A2B5- and NG2-positive cells after multiple passages (at passage 11) accompanied with a freeze-thawing cycle. Scale bar: 20 µm. ( H ) Quantification of marker-positive cells at passage 11. ( I ) Quantitative gene expression of OPC markers ( PDGFRα and SOX10 ). Scale bars: 10 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Student t -test.

Article Snippet: One day later, the cells were infected with a lentivirus encoding SOX10 (Addgene, clone ID number 45843, Watertown, MA, USA) and a lentivirus of reverse tetracycline-controlled transactivator ( rtTA ) (Addgene, clone ID number 20342) for doxycycline (DOX)-induced SOX10 expression and incubated for 14–16 h. Viral vectors and packaging were constructed as described elsewhere [ ].

Techniques: Derivative Assay, Gene Expression, Marker

Journal: Biology

Article Title: A Novel Vitronectin Peptide Facilitates Differentiation of Oligodendrocytes from Human Pluripotent Stem Cells (Synthetic ECM for Oligodendrocyte Differentiation)

doi: 10.3390/biology10121254

Figure Lengend Snippet: Differentiation of oligodendrocytes from hPSC-derived OPCs via SOX10 overexpression. ( A ) A schematic diagram for oligodendrocyte differentiation from hPSC-derived OPCs. ( B – D ) Time-course expression of OPC markers during differentiation. As differentiation proceeded, the transcript levels of OLIG2 and PDGFR-α gradually decreased ( C , D ) whereas that of SOX10 was maintained due to continued exogenous expression ( B ). ( E – G ) Time-course expression of OD markers during differentiation. Expression levels of all marker genes tested were upregulated by SOX10-induced differentiation and plateaued after day 8. ( H , I ) Representative image of O4- and MBP-positive cells induced by the overexpression of SOX10. Scale bars: 12.5 µm. ** p < 0.01, **** p < 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: One day later, the cells were infected with a lentivirus encoding SOX10 (Addgene, clone ID number 45843, Watertown, MA, USA) and a lentivirus of reverse tetracycline-controlled transactivator ( rtTA ) (Addgene, clone ID number 20342) for doxycycline (DOX)-induced SOX10 expression and incubated for 14–16 h. Viral vectors and packaging were constructed as described elsewhere [ ].

Techniques: Derivative Assay, Over Expression, Expressing, Marker

Journal: Biology

Article Title: A Novel Vitronectin Peptide Facilitates Differentiation of Oligodendrocytes from Human Pluripotent Stem Cells (Synthetic ECM for Oligodendrocyte Differentiation)

doi: 10.3390/biology10121254

Figure Lengend Snippet: Screening for ECM materials facilitating oligodendrocyte differentiation induced by SOX10. ( A – F ) Representative images of O4- and MBP-positive cells generated on the indicated matrix. White arrows are pointing to double-positive cells for O4 and MBP. ( G , H ) Quantification of the number of O4- or MBP-positive cells on day 12 after initiation of oligodendrocyte maturation on the indicated substrates. * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: One day later, the cells were infected with a lentivirus encoding SOX10 (Addgene, clone ID number 45843, Watertown, MA, USA) and a lentivirus of reverse tetracycline-controlled transactivator ( rtTA ) (Addgene, clone ID number 20342) for doxycycline (DOX)-induced SOX10 expression and incubated for 14–16 h. Viral vectors and packaging were constructed as described elsewhere [ ].

Techniques: Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: Establishing the covalently reactive peptide/protein pair SnoopTag and SnoopCatcher. (A) Spontaneous isopeptide bond formation between Lys and Asn, releasing ammonia. (B) Cartoon of splitting RrgA D4 domain [based on Protein Data Bank (PDB) ID code 2WW8] to make SnoopTag and SnoopCatcher. Reactive residues are in cyan. (C) SnoopTag-MBP reaction with SnoopCatcher, each at 10 µM, after 2 h at 25 °C analyzed by SDS/PAGE with Coomassie staining, alongside controls with Ala mutation of SnoopTag’s reactive Lys (KA) or SnoopCatcher’s reactive Asn (NA). (D) Isopeptide bond formation between SnoopTag peptide and SnoopCatcher shown by mass spectrometry. (E) Time course of SnoopTag reaction with a 1:1 or 2:1 ratio of SnoopCatcher to SnoopTag-MBP, tested as in C. (F) Time course of SnoopCatcher reaction with a 1:1, 2:1, or 4:1 ratio of SnoopTag-MBP to SnoopCatcher, tested as in C. Error bars are mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Mutagenesis, Mass Spectrometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: SnoopTag and SnoopCatcher reaction conditions. (A) Point mutations to make SnoopCatcher. From the C-terminal domain of RrgA in cartoon format (based on PDB ID code 2WW8), residues mutated are shown in space-fill with carbons in cyan (G842 was changed to T and D848 to G). The Lys, Asn, and Glu involved in isopeptide bond formation are shown in stick format in yellow and the rest of SnoopTag colored magenta. (B) Sample gel to test for quantitative reaction of SnoopTag. Shown are 10 µM SnoopTag-MBP and 20 µM SnoopCatcher alone or mixed for 32 min (triplicate samples) before SDS/PAGE with Coomassie staining. (C) Sample gel to test for quantitative reaction of SnoopCatcher. Shown are 10 µM SnoopCatcher and 40 µM SnoopTag-MBP alone or mixed for the indicated times (triplicate samples) before SDS/PAGE with Coomassie staining. (D) Buffer dependence of SnoopTag/SnoopCatcher reaction. We incubated 10 µM SnoopTag-MBP with 10 µM SnoopCatcher at pH 8.0 for 15 min at 25 °C in the indicated buffer and analyzed the samples by SDS/PAGE with Coomassie staining. (E) TMAO dependence of SnoopTag/SnoopCatcher reaction tested as in D. Error bars are all mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Incubation