Journal: The Journal of Biological Chemistry

Article Title: Non-muscle tropomyosins inhibit myosin-19 and dynamically localize to mitochondrially associated actin filaments

doi: 10.1016/j.jbc.2026.111377

Figure Lengend Snippet: Sequence and structural alignment of myosin Loop 4 region and expression of Myo19-3xIQ . A , primary structure alignment of Loop 4 region (boundary marked with dashed lines) of Myo19, Myo1C, Myo1B, Myh11 (smooth muscle myosin), and Myh10 (non-muscle myosin II B), motor domains. Charge state of residues highlighted in blue (positive) and red (negative). Magenta boxes represent predicted HL and HM helices that come before and after the Loop 4 region . The column of numbers is the net charge of Loop 4 for aligned myosin sequences. Arrows to the left of the chart indicate whether the myosin is inhibited or activated by the presence of Tpms. B , predicted structure of Myo19’s Loop 4 as well as its modeled electrostatic surface. C , aligned representation of predicted Myo19 Loop 4 if bound to actin ( cyan ) and Tpm3.1 (electrostatic surface). D , reported structure of rigor Myh11’s Loop 4 (PDB: 6BIH ) as well as its modeled electrostatic surface. E. Aligned representation of reported Myh11 Loop 4 if bound to actin ( cyan ), and Tpm3.1 (electrostatic surface). F , Coomassie-stained SDS-Page gel of final elution from FLAG affinity purification protocol containing the Myo19-3xIQ, coexpressed RLC12B and calmodulin light chains, and contaminating EIF-4B ( arrowhead ) protein that contains multiple FLAG-like repeats.

Article Snippet: Primary antibodies used with 4% paraformaldehyde and 4% sucrose fixation include Tpm3 (anti-Mouse, DSHB – CG3-S, 1:1000 dilution) and Hsp60 (anti-Chicken, Invitrogen – PA5-143571, 1:2000 dilution).

Techniques: Sequencing, Expressing, Staining, SDS Page, Affinity Purification

Journal: The Journal of Biological Chemistry

Article Title: Non-muscle tropomyosins inhibit myosin-19 and dynamically localize to mitochondrially associated actin filaments

doi: 10.1016/j.jbc.2026.111377

Figure Lengend Snippet: Both Tpm1.7 and Tpm3.1 show concentration dependent inhibition of Myo19-driven motility . A , representative images of surface-bound actin filaments at saturating concentrations of Tpm1.7/3.1 and 75 nM or 100 nM of Myo19-3xIQ. B , binary assessment of presence of surface bound filaments across a range of Myo19 concentrations. C , representative images of actin filaments bound to a Myo19-3xIQ coated surface at either 0 μM, 1 μM, or 3 μM of Tpm1.7/3.1. D , fraction of surface-bound filaments normalized to 0 μM Tpm1.7/3.1 control as a function of Tpm concentration. E , percentage of motile filaments normalized to 0 μM Tpm1.7/3.1 controls as a function of Tpm concentration showing all data points (shaded) and averages of binned data (solid points), diamond outlined points indicate no filaments were bound to the surface. F , average speed of motile surface-bound filaments across a range of Tpm1.7/3.1 concentrations, solid line represents the average speed of 0 μM Tpm control chambers and shaded region indicate ± 1 standard deviation.

Article Snippet: Primary antibodies used with 4% paraformaldehyde and 4% sucrose fixation include Tpm3 (anti-Mouse, DSHB – CG3-S, 1:1000 dilution) and Hsp60 (anti-Chicken, Invitrogen – PA5-143571, 1:2000 dilution).

Techniques: Concentration Assay, Inhibition, Control, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Non-muscle tropomyosins inhibit myosin-19 and dynamically localize to mitochondrially associated actin filaments

doi: 10.1016/j.jbc.2026.111377

Figure Lengend Snippet: Myo19-Tpm3.1 and Tpms compete for actin binding . A , Time-lapse images depicting smooth muscle myosin–driven motility of rhodamine–phalloidin–stabilized actin filaments decorated with GFP–Tpm3.1 (5 μm scale bar). B , left . Still frame of rigor Myo19-3xIQ coated surfaces in the presence of GFP-Tpm3.1 coated rhodamine phalloidin stabilized actin filaments (5 μm scale bar). B , right . Series of time-lapse images showing Myo19-3xIQ driven gliding of rhodamine phalloidin stabilized actin filaments in the presence of ATP and the lack of corresponding GFP-Tpm3.1 signal (5 μm scale bar). C , quantification of GFP-Tpm3.1 signal that overlaps with rhodamine phalloidin signal for Myo19-3xIQ with or without ATP (Mean ± 1 Stdev.). D , graphical representation of TIRF results where GFP-Tpm3.1 binds to actin in the absence of motor, competes for binding with Myo19-3xIQ, and bind simultaneously with smooth muscle myosin.

Article Snippet: Primary antibodies used with 4% paraformaldehyde and 4% sucrose fixation include Tpm3 (anti-Mouse, DSHB – CG3-S, 1:1000 dilution) and Hsp60 (anti-Chicken, Invitrogen – PA5-143571, 1:2000 dilution).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Non-muscle tropomyosins inhibit myosin-19 and dynamically localize to mitochondrially associated actin filaments

doi: 10.1016/j.jbc.2026.111377

Figure Lengend Snippet: Endogenous Tpm3 is enriched on mitochondrial-associated actin filaments . A , Western blot analysis ( Top ) of endogenous expression of Tpm1.6/1.7 and Tpm3 in HeLa lysates, dashed line indicates predicted size od Tpm1.6/1.7, bottom images are corresponind total protein stains. B and C , representative micrograph of fixed HeLa cells with F-actin labeled using rhodamine-phalloidin ( cyan ), and Tpm3 ( magenta ) and mitochondria ( yellow ) labeled using corresponding antibodies (5 μm scale bar). D , inset images showing the localization of actin and Tpm3 near mitochondria when the actin wave is (i) or is not (ii) present (2 μm scale bar). E , quantification of endogenous Tpm3 fluorescence signal overlapping with mitochondria that are or are not surrounded by the actin wave (Mean ± 1 Stdev.).

Article Snippet: Primary antibodies used with 4% paraformaldehyde and 4% sucrose fixation include Tpm3 (anti-Mouse, DSHB – CG3-S, 1:1000 dilution) and Hsp60 (anti-Chicken, Invitrogen – PA5-143571, 1:2000 dilution).

Techniques: Western Blot, Expressing, Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Non-muscle tropomyosins inhibit myosin-19 and dynamically localize to mitochondrially associated actin filaments

doi: 10.1016/j.jbc.2026.111377

Figure Lengend Snippet: A Tpm3.1 wave traverses the cell concurrently as the actin wave . A and B , representative micrograph images showing full frame merge along with inset merge and individual channel images from long-term timelapse videos of HeLa cells overexpressing LifeAct mScarlett ( cyan ), GFP-Tpm3.1 ( magenta ), and mitochondrial labeled with mitoTracker DeepRed ( yellow ) at an initial time-point and 120 and 240 s later (5 μm scale bar). C , individual fluorescence trace of LifeAct-mScarlett ( blue ) and GFP-Tpm3.1 ( red ) as it traverses a region of the cell. D , ensemble averages of many individual traces aligned at a point prior to actin-wave fluorescence intensity increase show simultaneous appearance of LifeAct-mScarlet and GFP-Tpm3.1 signal (Mean ± 1 Stdev.). Shaded region denotes timepoints where the Mann–Whitney U test determined there was significant difference in the rate of dispersion. E , schematic depicting the role of Tpm3.1 in the actin wave determining which population of actin Myo19 can or cannot interact with.

Article Snippet: Primary antibodies used with 4% paraformaldehyde and 4% sucrose fixation include Tpm3 (anti-Mouse, DSHB – CG3-S, 1:1000 dilution) and Hsp60 (anti-Chicken, Invitrogen – PA5-143571, 1:2000 dilution).

Techniques: Labeling, Fluorescence, MANN-WHITNEY, Dispersion