Actin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin

ABSTRACT

Actin filaments are dynamic polymers whose length depends on regulated monomer association and dissociation at their ends. Actin barbed‐end dynamics are relatively better understood, primarily due to the approximately tenfold faster subunit on/off rates at barbed versus pointed ends. We present experimental approaches to selectively assay actin pointed‐end regulation using bulk biochemistry, single filament imaging, and live cell microscopy with an emphasis on tropomodulins (Tmods), a conserved family of eukaryotic proteins that specifically cap pointed ends. Average pointed‐end assembly/disassembly rates are measured in bulk solution using pyrene‐labeled actin and barbed end‐capping protein CapZ. Direct rate measurements of individual pointed ends are performed via microfluidic‐assisted total internal reflection fluorescence microscopy (mf‐TIRF). Actin pointed‐end dynamics in living cells are examined in striated muscle cells expressing fluorescent actin, where the regular arrays of 1‐ to 2‐μm‐long actin filaments in sarcomeres enable visualization of filament pointed and barbed ends. These assays will also help advance our understanding of other pointed end regulators, including cyclase‐associated protein and leiomodins, which have been implicated in filament stabilization, disassembly, and elongation. This work is relevant to the musculoskeletal field, where precise regulation of filament lengths is particularly critical for sarcomere organization and striated muscle contraction.

1. Introduction

Actin filament assembly and dynamics is fundamental to cell behaviors—whether it be assuming a specific shape, moving in a specific direction, producing mechanical force, or maintaining connections with neighbors in tissues. In musculoskeletal cells and tissues, the actin cytoskeleton plays important roles, including the actin filament cortex that maintains differentiated chondrocytes in cartilage (Park et al. 2008; Parreno et al. 2017; Woods et al. 2005), the actin filament bundles in osteocyte dendritic processes that play a role in mechanotransduction in bone (Guerra et al. 2024; You et al. 2001, 2004), and the actin (thin) filaments of sarcomeres in striated muscle cells that interact with myosin to generate contractile forces (Clark et al. 2002; Gokhin and Fowler 2011; Henderson et al. 2017). In striated muscle, where contractile force is produced by myosin crossbridge interactions with thin filaments, the amount of active force depends on the degree of actin and myosin filament overlap. This in turn depends on precise thin filament lengths which are muscle‐specific and determine the operating range of lengths for individual muscles tailored to their physiological functions (Gokhin and Fowler 2011, 2013; Littlefield and Fowler 1998, 2008; Szikora et al. 2022). Alterations of thin filament lengths in development, aging, pathological, or disease conditions have profound effects on muscle contractile force generation and physiological function (Clark et al. 2002; Henderson et al. 2017).

Actin filaments are dynamic polymers that depend on regulated monomer association and dissociation at each end, with subunit on/off rates ~10X greater at fast‐growing (barbed) ends than at slow‐growing (pointed) ends (Goode et al. 2023; Lappalainen et al. 2022; Pollard 1986). The kinetics of actin incorporation at filament ends are regulated, in part, by protein caps, which bind to filament ends and inhibit monomer association and dissociation. While there is a host of proteins that cap barbed ends, at pointed ends, high affinity cappers are the Arp2/3 complex (Mullins et al. 1998) and the tropomodulins (Tmods) (Weber et al. 1994). Here, we will focus on the Tmods, which include four canonical ~40 kD Tmod isoforms (Tmods1‐4), with tissue‐specific and developmentally regulated patterns of expression in vertebrates, worms, and flies (Yamashiro et al. 2012). Tmods also bind directly to tropomyosins (Tpms) (Fowler 1987), which cooperate synergistically with Tmods to cap actin pointed ends and stabilize filaments in muscle and nonmuscle cells (Gokhin and Fowler 2011; Parreno and Fowler 2018; Yamashiro et al. 2012). Leiomodins (Lmods 1–3) are larger Tmod‐related proteins in striated and smooth muscles, which bind tropomyosins (Tpms) and nucleate actin filament assembly in vitro (Chereau et al. 2008) and are proposed to promote elongation from thin filament pointed‐ends in vivo (Tolkatchev et al. 2020), although the molecular mechanisms are unclear. For a comprehensive review, see (Fowler and Dominguez 2017).

In contrast to the barbed filament end, which has been studied extensively, actin subunit filament assembly/disassembly at the pointed filament end is less well understood. This is principally due to challenges in studying the infrequent events at pointed ends as compared to the frequent events at barbed ends. In bulk polymerization assays where both filament ends can polymerize or depolymerize, readout signals are dominated by rapid subunit addition or loss at barbed ends, so that relatively less frequent events at pointed ends are not detected. On the other hand, in single filament assays using total internal reflection fluorescence (TIRF) microscopy, spontaneous actin nucleation and barbed end elongation of new filaments can make it challenging to study events at pointed ends since actin monomer concentrations needed for subunit addition to pointed ends are above the actin critical concentration (Fujiwara et al. 2007; Lappalainen et al. 2022). In living cells, the utilization of fluorescent actin probes has enabled investigations of actin dynamics, but again, signals are dominated by the frequent events at barbed ends. Moreover, not only does irregular actin filament network organization and variable filament lengths typically make it impossible to identify signals at pointed ends as distinct from barbed ends (Svitkina 2018), but actin monomers in cells are in majority bound to the protein profilin that inhibits their addition to the pointed end (Carlier and Shekhar 2017; Kaiser et al. 1999).

Here, we present detailed methods to selectively assay actin assembly and disassembly at pointed ends using a variety of complementary approaches. In Section 2, we present bulk actin polymerization assays using fluorescent‐labeled actin, in which average rates of pointed end assembly/disassembly are measured by silencing barbed ends with capping proteins such as gelsolin or CapZ. In Section 3, we present a microfluidic flow approach with TIRF microscopy (mf‐TIRF) that eliminates spontaneous formation of actin oligomers, enabling direct rate measurements of individual filament pointed ends and investigation of heterogeneities in filament assembly. In Section 4 we present methods using cultured striated muscle cells and fluorescent‐labeled actin to selectively measure actin dynamics at pointed and barbed ends. We focus our methods on the Tmod family of pointed end capping proteins, a conserved family of eukaryotic proteins known to specifically cap pointed ends in both muscle and non‐muscle cells. These assays will also advance our understanding of the expanding ecosystem of other pointed end regulators, comprising actin stabilization factors (e.g., Tpms), actin pointed end disassembly factors (e.g., cofilins, Cyclase‐associated protein 2, (Cap2)), and demonstrated (e.g., bacterial effector, VopF) (Kudryashova et al. 2022) or proposed pointed end elongation factors (Lmods) (Tolkatchev et al. 2020). For recent reviews on actin pointed end regulation, see (Fowler and Dominguez 2017; Goode et al. 2023; Iwanski et al. 2021).

2. Bulk Actin Polymerization Assays

2.1. General Considerations

Actin purified from rabbit or chicken skeletal muscle (α‐actin) is most commonly used in actin filament (F‐actin) polymerization assays. If nonmuscle actin (β/γ actin) is desired, actin is commonly purified from platelets or other tissues such as brain or liver (Okamoto et al. 1983; Rosenberg et al. 1981; Schafer et al. 1998). More recently, various recombinant actins have also been expressed and purified from Pichia pastoris (Hatano et al. 2018). While purification of actin monomers (G‐actin) is relatively straightforward (Section 2.1, below), actin oligomers and aggregates in G‐actin preparations can act as nuclei for polymerization, interfering with specific detection of pointed‐end assembly. It is desirable that they be removed from the G‐actin prior to assays, which can be accomplished with gel filtration chromatography (MacLean‐Fletcher and Pollard 1980). It should be noted that commercially available lyophilized actin tends to contain actin aggregates after resuspension; thus, caution is required in assay design and interpretation. The presence of actin oligomers or aggregates can be evaluated in a control experiment by measuring polymerization kinetics of G‐actin to confirm the presence of a long lag phase (> 1 min for 10 μM actin) before enough nuclei accumulate to observe initiation of fast polymerization in the elongation phase. In addition, the ability of actin to polymerize should be confirmed by measuring the critical concentration of actin, as old or denatured G‐actin tends not to polymerize.

Polymerization or depolymerization at F‐actin pointed ends is typically measured spectrofluorometrically using pyrene‐labeled actin (pyrene‐actin) monomers with barbed‐end capped F‐actin seeds. Pyrene‐actin monomers exhibit a ~25‐fold increase in fluorescence upon polymerization to F‐actin, which enables measuring the kinetics of actin assembly/disassembly with high sensitivity (Kouyama and Mihashi 1981). It should be noted that since pyrene assays only measure the total amount of F‐actin in the sample, if a protein promotes actin assembly in a sample without F‐actin seeds, it is unclear whether it promotes the nucleation or elongation step. Further, the presence of contaminating actin oligomers in the G‐actin solution can accelerate elongation, resulting in increased F‐actin (indicated by increased pyrene‐actin fluorescence), but reducing the experimenter's ability to detect an increase in F‐actin due to the inhibition of pointed end elongation. We present a protocol for preparing pyrene‐labeled G‐actin from rabbit skeletal muscle acetone powder in Section 2.2.

2.2. Preparation of Proteins

2.2.1. Preparation of Pyrene‐Labeled Actin

A method for the large‐scale purification of actin from rabbit skeletal muscle has been widely used for several decades (Pardee and Spudich 1982; Spudich and Watt 1971). Rabbit skeletal muscle acetone powder from PelFreez Biologicals (Rogers, AR) is commercially available and can be used as a source of G‐actin for biochemical assays (Ono et al. 2011). In this section, we present a protocol for extracting actin from rabbit skeletal muscle acetone powder, labeling it with N‐(1‐pyrene) iodoacetoamide, and purifying the pyrene‐labeled G‐actin. If the labeling step is omitted, unlabeled G‐actin can be purified using the same protocol.

For preparation and storage of pyrene‐actin, we follow the method of Cooper et al. (1983), in which N‐(1‐pyrene) iodoacetoamide is used to label actin at Cys374. It is important to note that modification of actin at Cys374 by conjugation with pyrene reduces the affinity of profilin for G‐actin (Malm 1984) and reduces the affinity of myosin for F‐actin by about half (Taylor 1991). Additionally, binding of myosin and cofilin quenches the fluorescence of pyrene‐labeled F‐actin (Carlier et al. 1997; Kouyama and Mihashi 1981). Therefore, in experiments in which these actin regulatory proteins are present, actin assembly/disassembly at pointed ends should also be analyzed by methods that use actin without pyrene labeling.

2.2.1.1. Required Materials

• Rabbit skeletal muscle acetone powder: Available from PelFreez Biologicals (Rogers, AR).

• G‐buffer: 2 mM Tris–HCl, 0.2 mM CaCl₂, 0.1 mM adenosine triphosphate (ATP), 1 mM dithiothreitol (DTT), pH 8.0 (4°C)

• 10 × Polymerizing solution: 500 mM KCl, 20 mM MgCl₂, 2 mM EGTA

• N‐(1‐pyrene) iodoacetoamide (Molecular Probes)

• F‐buffer (−DTT): 20 mM HEPES‐KOH, 0.1 M KCl, 2 mM MgCl₂, 0.2 mM ATP, pH 7.5

2.2.1.2. Procedure for Pyrenyl Labeling of Actin

1. G‐actin is prepared from an acetone powder according to the method of Spudich and Watt (Spudich and Watt 1971). Five grams of acetone powder are placed in a beaker on ice and 150 mL of cold G‐buffer is slowly added while gently stirring with a glass rod to thoroughly wet the powder. Let sit on ice for 30 min, stirring gently once every 10 min.

2. The extract is separated from the acetone powder residue by, for example, suction filtration through a Buchner funnel set with filter paper, or by centrifugation. Then, remaining debris from the extract is removed by further filtration using a syringe filter with a 0.45‐μm membrane.

3. G‐actin is polymerized by adding a 1/9 volume of 10 × polymerizing solution and incubated for 2 h at 25°C.

4. 5 M NaCl is added to a final concentration of 0.6 M to dissociate tropomyosin from F‐actin and incubated for 1 h at 25°C.

5. Pellet F‐actin by centrifugation at 100,000 × g for 1 h at 4°C.

6. The F‐actin pellet is resuspended by adding 2 mL of cold F‐buffer (minus DTT) to ultracentrifuge tubes. The resuspended F‐actin is transferred into a new 15‐mL tube and the F‐actin solution is dispersed by sonicating with a probe sonicator while placing the tube on ice.

7. The F‐actin solution is next dialysed against 500 mL of F‐buffer (minus DTT). It is necessary to remove DTT completely as it prevents the pyrene labeling reaction.

8. Measure protein concentration of the F‐actin solution with methods such as the Bicinchoninic acid (BCA) protein assay.

Note: F‐actin solution may be sticky. Be careful to measure the volume accurately.

• 9Dilute F‐actin to 1 mg/mL (~23.8 μM) with F‐buffer (minus DTT).
• 10Prepare a 20× stock solution of 38 mM N‐(1‐pyrene) iodoacetamide (NPI) dissolved in dimethylformamide.
• 11While mixing, add a 1/19 volume of the stock solution to bring the NPI to 8‐fold over the molar concentration of F‐actin, i.e., NPI final concentration of 190 μM.

Note: The solution becomes white after mixing due to the low solubility of NPI.

• 12Wrap the tube with aluminum foil. Incubate overnight on a rotator at 4°C.

Note: Since pyrene can be photobleached, pyrene‐actin samples should be shielded from light. When mixing the sample on a rotator, be careful not to let bubbles form in the sample.

• 13Add DTT to a final concentration of 5 mM to stop the labeling reaction.
• 14Centrifuge for 10 min at 10,000 × g to remove insoluble NPI. Transfer the supernatant to ultracentrifuge tubes.
• 15Pellet F‐actin by centrifugation at 100,000 × g for 1 h at 4°C.
• 16Resuspend the pellet by homogenizing or sonicating in 2 mL of cold G‐buffer and dialyze overnight at 4°C against 500 mL of G‐buffer in a beaker wrapped with aluminum foil to prevent photobleaching. During dialysis, change the G‐buffer at least once.
• 17Centrifuge the pyrene‐actin solution at 100,000 × g for 1 h to remove aggregates. Collect the top two‐thirds of the supernatant for further purification on a gel filtration column in the next step.
• 18Purify labeled G‐actin by gel filtration using a Superdex 200 pg HiLoad 16/60 column (16 × 600 mm; GE Healthcare). To perform gel filtration chromatography, we have used an AKTA purifier or AKTAprime plus liquid chromatography system (GE Healthcare). Run G‐buffer through the column and collect 2 mL fractions in the dark at 4°C. The pyrene‐actin fractions at or past the peak should be used for further experiments, as they will be most enriched for actin monomers, with reduced contamination by capping protein, actin dimers, trimers, or other oligomers in the leading edge.
• 19Determine the concentration of pyrene by measuring the absorbance at 344 nm in the purified pyrene‐actin solution, and the concentration of total G‐actin including pyrene‐labeled G‐actin by measuring the absorbance at 290 nm and at 344 nm. Calculate the extent of labeling by dividing the concentration of pyrene by the concentration of total G‐actin. G‐actin thus prepared is usually ~90% labeled.

Pyrene concentration: [pyrene, μM] = Abs₃₄₄/2.2 × 10⁴ (M⁻¹).

G‐actin concentration including pyrene‐actin:

[G‐actin, μM] = (Abs₂₉₀‐Abs₃₄₄ × 0.127)/2.66 × 10⁴ (M⁻¹)

• 20Purified actin monomers in G‐buffer should be stored on ice and dialyzed against G‐buffer containing fresh ATP and DTT every 4–5 days for use within 2 weeks (Xu et al. 1998). Pyrene‐actin should be stored in the dark. If long‐term storage is required, unlabeled actin or pyrene‐actin can be divided into small aliquots (~25–50 μL), flash frozen in liquid N₂, and stored at −80°C, but care is necessary to prevent actin protein denaturation or aggregation due to freezing or thawing (Xu et al. 1998). Thus, G‐actin aliquots should be thawed quickly and ultracentrifuged to remove any aggregates (e.g., 30 min, 450,000 × g) before performing polymerization assays (Fowler et al. 2003). In addition, as explained above (IIA), the presence of actin oligomers or aggregates can be evaluated in a control experiment by measuring polymerization kinetics of G‐actin to confirm the presence of a long lag phase (> 1 min for 10 μM actin) before the elongation phase.

2.2.2. Preparation of Tmods

Native Tmod1 protein was originally purified from isolated erythrocyte membranes (Fowler 1987, 1990) which are highly enriched for filament ends due to the short F‐actin lengths (40 nm), each capped by a Tmod1 molecule (Tmod1/actin subunit = 1/16) (Fowler 1990). However, purification of native Tmod1 in milligram quantities is an arduous, many‐day procedure, starting with several units of whole blood to provide several hundred milliliters of packed erythrocyte membranes as a starting material, followed by selective extractions, anion exchange column chromatography, and sucrose gradient centrifugation steps, with a final yield of 1–2 mg Tmod1 (Fowler 1990). Therefore, we have used recombinant Tmods fused at their N‐terminus to glutathione S‐transferase (GST), that were expressed in the pGEX‐KG vector in BL21 Escherichia coli (Fischer et al. 2006; Fowler et al. 2003; Weber et al. 1994; Yamashiro et al. 2014, 2010). In most biochemical experiments, we remove GST‐tags by thrombin cleavage after affinity purification of GST‐Tmod and further purify Tmod proteins using RESOURCE Q anion exchange chromatography. Tmods purified in this manner exhibit F‐actin pointed‐end capping activity (Fowler et al. 2003; Weber et al. 1994), Tpm binding, and Tpm‐dependent, high‐affinity F‐actin pointed‐end capping activity (Fowler et al. 2003; Lewis et al. 2014; Weber et al. 1994; Yamashiro et al. 2014). Some Tmod isoforms also have nucleating (Yamashiro et al. 2010) as well as G‐actin‐binding activities (Fischer et al. 2006; Yamashiro et al. 2010). Comparisons of native Tmod1 purified from human red blood cells with recombinant Tmod1 prepared from GST‐Tmod1 fusion protein have not revealed functional differences with respect to actin pointed‐end capping or Tpm binding. Recombinant His‐tagged Tmods (Kostyukova et al. 2000; Moroz et al. 2013; Vasilescu et al. 2024) or recombinant Tmod expressed as an intein‐fusion protein precursor (Rao et al. 2014) have also been used by others in Tpm binding and actin pointed‐end polymerization assays. Here, we describe a protocol for purification of recombinant Tmods from E. coli cells expressing GST‐Tmod proteins.

2.2.2.1. Required Materials

• Chemically competent BL21 (DE3) E. coli cells

• Luria Broth (LB) with 50 μg/mL ampicillin

• Phosphate‐buffered Saline (PBS) lysis Buffer: PBS containing protease inhibitors

• Glutathione Sepharose (GE Healthcare)

• Glutathione S‐Transferase (GST)

• Thrombin cleavage buffer: 100 mM NaCl, 20 mM Tris–HCl pH 8.0, 2.5 mM CaCl₂, 1 mM DTT

• Biotinylated Thrombin (Novagen)

• Streptavidin agarose (Novagen)

• RESOURCE Q anion exchange chromatography column (6 mL; GE Healthcare)

• Buffer A: 20 mM Tris–HCl pH 8.0, 0.2 mM DTT

• F‐Buffer: 20 mM Hepes (pH 7.5), 2 mM MgCl₂, 0.1 M KCl, 1 mM DTT

2.2.2.2. Purification of Recombinant Tmods

1. Transform E. coli BL21(DE3) cells with a plasmid for the expression of GST‐fused Tmod.

2. Grow transformed cells in 2 L of Luria Broth with 50 μg/mL ampicillin to log phase at 37°C.

3. Cool down by soaking the flask in tap water. Induce protein expression at 25°C for 4 h by the addition of 0.1 mM IPTG.

4. Harvest cells by centrifugation at 11,200 × g for 15 min. The cell pellets can either be used immediately or stored at −80°C for up to several months.

5. Add 5 times the volume of ice‐cold PBS lysis buffer to the E. coli cell pellet and resuspend the pellet in the buffer.

   Note: We harvest 2 L of E. coli culture and collect ~10 mL bacterial pellet. All Tmods1‐4 express well in E. coli, with typical yields of 0.3–0.8 mg/L of culture.

6. Disrupt bacterial cells to extract proteins by appropriate methods (e.g., sonication, French press). In the case of sonication, lyse cells using a probe sonicator by sonicating 5 times with 30‐s on–off alternating cycles. Keep cells on ice throughout the process.

7. Centrifuge the lysate at 10,000 × g for 30 min at 4°C.

8. Transfer the supernatant to clean 50 mL conical tubes. The supernatant fraction that is the bacterial extract contains soluble GST‐Tmod protein.

9. Purify GST‐Tmod protein from the bacterial extract using commercial glutathione resin according to the manufacturer's instructions, eluting the GST‐Tmod protein with excess glutathione. Glutathione resin should be used in amounts capable of binding ≥ 60 mg of GST.

10. Dialyze the eluted GST‐Tmod against 500 mL of Thrombin cleavage buffer (100 mM NaCl, 20 mM Tris–HCl pH 8.0, 2.5 mM CaCl₂, 1 mM DTT), changing the dialysis buffer once.

11. Measure the protein concentration of the GST‐Tmod fraction with protein quantification assays compatible with reducing agents, including DTT.

12. Add biotinylated thrombin (Novagen) to the GST‐Tmod fraction at 0.5 units per 1 mg GST‐Tmod protein. Incubate for 2 h at 25°C.

13. To stop the cleavage reaction, add Streptavidin agarose beads to the biotinylated thrombin and GST/Tmod mixture. Incubate for 20 min on a rotator at 25°C. Remove the Streptavidin agarose beads by filtration of the protein solution using a syringe filter with a low protein‐binding 0.22 μm PVDF membrane (Millipore) then place the protein sample on ice.

14. During thrombin treatment, prepare for the RESOURCE Q chromatography step. To perform the RESOURCE Q chromatography, we have used an AKTA liquid chromatography system (GE Healthcare). Equilibrate the column with Buffer A (20 mM Tris–HCl pH 8.0, 0.2 mM DTT at 4°C).

15. Add an equal volume of Buffer A to the GST and Tmod protein sample to reduce the NaCl concentration in the sample. The NaCl concentration will be reduced from 100 to 50 mM. Eliminate debris by filtration with a 0.22 μm membrane, and then apply the protein sample to the column.

16. Wash the column with 5 column volumes (CVs) of Buffer A.

17. Elute proteins from the column with a linear NaCl gradient (0.05–0.4 M) for 40 CV and collect fractions at 4°C.

18. Run SDS‐PAGE of collected fractions to identify fractions containing purified Tmod (~40 kDa) as a single band.

19. Dialyze the pooled fractions into F‐buffer (Section 2.1).

20. Divide the purified Tmod sample into ~50–200 μL aliquots in microfuge tubes, freeze quickly in liquid N₂, and store at −80°C.

    Note: The extinction coefficients (Abs _0.1% ²⁸⁰ ) of Tmods1‐4 are as follows: Tmod1 (human), 0.532; Tmod1 (chicken), 0.353; Tmod2 (rat), 0.148; Tmod3 (mouse), 0.191; Tmod3 (human), 0.187; Tmod4 (mouse), 0.517; Tmod4 (chicken), 0.312. Characteristics and activities of recombinant Tmods 1–4 are summarized in Table 1 in our previous study (Yamashiro et al. 2010).

2.2.3. Preparation of Tropomyosins (Tpms)

Tpms form rod‐like alpha‐helical coiled‐coil dimers, with more than 40 isoforms of Tpms expressed from 4 different genes in different cells and tissues in vertebrates (Gunning et al. 2015; Hardeman et al. 2020). Tpms contain 9 alternately spliced exons and can be grouped into higher molecular weight “long” isoforms containing an exon 1a and 2b, and “short” isoforms with an exon 1b and no exon 2. The long Tpm isoforms are abundant in striated and smooth muscles, where they are associated with the thin filaments as mixed populations of heterodimers and/or homodimers depending on the muscle, and can be purified from these tissues (Lau et al. 1985; Smillie 1982). The short Tpm isoforms are abundant in non‐muscle cells, with most cells expressing both short and long isoforms (Gunning et al. 2015). Thus, preparation of recombinant Tpms allows isolation and study of individual isoform homodimers or heterodimers (Coulton et al. 2006; Kalyva et al. 2012; Kis‐Bicskei et al. 2013; Maytum et al. 2004). However, an important consideration for pointed end capping studies is that native eukaryotic Tpms are acetylated at the N‐terminus (Perry 2001), while bacterially expressed Tpms are not. This post‐translational modification affects the F‐actin binding properties of the long but not short Tpms (Kostyukova and Hitchcock‐DeGregori 2004; Moraczewska et al. 1999), as well as their Tmod‐binding ability (Greenfield and Fowler 2002; Kostyukova and Hitchcock‐DeGregori 2004), making bacterially expressed recombinant long Tpms unsuitable for studies of pointed end capping by Tmods. Therefore, our work with striated muscle Tpms has relied upon native Tpm isoforms purified from muscle tissue sources (Almenar‐Queralt et al. 1999; Babcock and Fowler 1994; Weber et al. 1994). Since the lack of N‐terminal acetylation of short Tpms does not appear to affect Tmod binding (Greenfield and Fowler 2002; Kostyukova and Hitchcock‐DeGregori 2004), we have utilized recombinant bacterially expressed Tpms for pointed end capping assays with Tmod (Lewis et al. 2014; Yamashiro et al. 2014), and this method is presented below.

2.2.3.1. Required Materials

• Chemically competent BL21 (DE3) E. coli cells.

• Luria Broth (LB).

• Lysis buffer—20 mM Tris pH 7.5, 0.5 M NaCl, 5 mM MgCl₂, protease inhibitors (0.5 μM each of pepstatin A, antipain, leupeptin, aprotinin, and chymostatin). Titrate pH at 4°C.

• 0.3 M HCl solution

• Buffer A—20 mM HEPES pH 6.8, 50 mM NaCl and 0.5 mM DTT

• Buffer B—20 mM HEPES pH 6.8, 1 M NaCl, 1 mM DTT

• Gel filtration buffer—20 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT, and 2 mM MgCl₂. Titrate pH at 4°C.

• Tip sonicator

• HiTrap Q HP 5 mL ion exchange column (Cytiva, 17,115,401)

• Centrifugal concentrator (Sartorius, VS0602)

• Superdex 75 Increase 10/300 (Cytiva, 29,148,721)

• Fast protein liquid chromatography (FPLC) system (Cytiva, ÄKTA pure 25 L)

2.2.3.2. Procedure

1. Transform E. coli BL21(DE3) cells with a plasmid for the expression of an untagged short Tpm isoform (e.g., Tpm1.9, Tpm3.1, Tpm4.2, etc.) (Geeves et al. 2015).

2. Grow transformed cells in 1 L of Luria Broth to log phase at 37°C.

3. Induce protein expression overnight at 18°C by the addition of 1 mM IPTG.

4. Harvest cells by centrifugation at 11,200 × g for 15 min. The cell pellets can either be used immediately or stored at −80°C for later use.

5. Resuspend the cell pellet in 30 mL of lysis buffer.

6. Lyse cells using a probe sonicator by sonicating 5 times with 30‐s on–off alternating cycles. Keep cells on ice throughout the process.

7. Incubate the cell lysate for 10 min at 80°C using a water bath.

8. Cool the cell lysate for 10 min in the −20°C freezer. Alternatively, the cell lysate can also be cooled in an ice/water bath.

9. Collect cell debris by centrifuging the lysate for 20 min at 40,000 × g at 4°C.

10. Collect the supernatant and discard the pellet.

11. Isoelectrically precipitate the proteins by dropwise addition of 0.3 M HCl until the pH reaches 4.5. Continue mixing throughout using a magnetic stir bar.

12. Collect the precipitated proteins by centrifuging at 18,000 × g for 15 min at 4°C.

13. Discard the supernatant and resuspend the pellet in 20 mL of 100 mM Tris, pH 7.5, 0.5 M NaCl, 5 mM MgCl₂, and 1 mM DTT.

14. Repeat isoelectric precipitation by dropwise addition of 0.3 M HCl until the pH reaches 4.5. Continue mixing throughout using a magnetic stir bar.

15. Centrifuge at 18,000 × g for 15 min at 4°C.

16. Resuspend the pellet in 20 mL of buffer A. Dialyze the solution overnight at 4°C against buffer A.

17. Next morning, clarify the protein at 18,000 × g for 30 min at 4°C. Discard the pellet and keep the supernatant.

18. Connect a 5 mL HiTrap Q HP column to the FPLC system and equilibrate the column in the dialysis buffer.

19. Load the protein onto the HiTrap Q HP column.

20. Elute the bound protein with a linear gradient of 0%–50% buffer B spread out over 50 mL of elution buffer. Collect 1 mL fractions.

21. Identify elution fractions containing Tpm using gel electrophoresis.

22. Pool peak fractions containing Tpm and concentrate to < 500 μL using a centrifugal concentrator.

23. Equilibrate Superdex 75 Increase 10/300 column with gel‐filtration buffer.

24. Load the protein on the column and collect fractions. Identify fractions containing Tpm using gel electrophoresis.

25. Pool peak fractions containing pure Tpm and measure the concentration using either gel‐densitometry or a spectrometer (Extinction coefficients determined by Expasy ProtParam tool on Expasy website). Aliquot and flash‐freeze the protein in liquid N₂. Store aliquots in an −80°C freezer until further use.

2.3. Assays for Pointed End Assembly/Disassembly

2.3.1. General Considerations

Bulk pointed‐end assembly/disassembly is measured using pyrene‐actin to monitor polymerization or depolymerization from pointed ends of F‐actin seeds capped at their barbed ends (Figure 1A). Barbed‐end (B‐end) capped F‐actin seeds are prepared prior to the addition of Tmods by copolymerization with capping protein (CP, or CapZ), which binds barbed ends tightly and blocks association/dissociation of actin monomers (Maun et al. 1996; Schafer et al. 1996). F‐actin seeds with varying average lengths can be prepared by varying the ratio of CapZ to actin (e.g., 1:50–1:100). It should be noted that long B‐end capped filaments may break and reveal new barbed ends, which can mask the effects of Tmod on pointed end assembly/disassembly. To avoid shearing of F‐actin seeds by pipetting, reduce the frequency of pipetting and use pipet tips with cut‐off tips. Gelsolin can also be used to prepare B‐end capped F‐actin seeds (Fowler et al. 2003; Weber et al. 1994; Yamashiro et al. 2010), but gelsolin‐actin binding can be unstable under some conditions, resulting in free barbed ends that interfere with the detection of pointed‐end actin assembly/disassembly (Rao et al. 2014; Vasilescu et al. 2024). We describe below a protocol to assay the pointed‐end capping activity of Tmods in the presence of Tpm. Tmod1 caps the pointed ends of Tpm‐decorated F‐actin with an affinity more than 1000‐fold greater than for bare actin pointed ends (K _d ~0.2 μM for pure actin and K _d in the nM‐to‐pM range for Tpm‐decorated F‐actin) (Kostyukova and Hitchcock‐DeGregori 2004; Weber et al. 1994, 1999). If Tpm is omitted, the same protocol can be used to examine Tpm‐independent effects of Tmods on pointed end assembly and disassembly.

FIGURE 1.

(A) Schematic diagrams of bulk P‐end assembly assays. Upper: Elongation of a B‐end capped F‐actin seed occurs at the P‐end when G‐actin > 0.6 μM. Lower: When Tmod caps the P‐end of a B‐end capped F‐actin seed, actin assembly at the P‐end is blocked. Tpm enhances Tmod's P‐end capping activity. (B) An example of kinetic measurements of pyrene‐actin polymerization with CapZ‐capped F‐actin seeds (CapZ:actin = 1:100). The data show actin polymerization in the presence or absence of CapZ‐capped F‐actin seeds, Tpm (TM5NM1), Tmod1, and Tmod3 as indicated in the figure. (C) An example of P‐end capping activity analysis of Tmod based on pyrene‐actin polymerization kinetics shown in B. P‐end capping activity of Tmod1 and Tmod3 was evaluated by measuring their ability to inhibit the initial elongation rates from the P‐ends of B‐end capped F‐actin seeds. The initial elongation rates were measured from the slopes of the polymerization traces over the first 1 min. The P‐end capping efficiencies of Tmod for TM5NM1‐coated F‐actin seeds were calculated using the formula shown in C. The data show that Tmod1 and Tmod3 exhibit similar P‐end capping efficiencies in the presence of TM5NM1. Data shown are mean ± S.D. of three experiments. Modified from (Yamashiro et al. 2014). BE, barbed end; PE, pointed end.

2.3.2. Preparation of CapZ‐Capped F‐Actin Seeds

Bacterially expressed recombinant chicken skeletal muscle CapZ α, β heterodimer is prepared as described (Soeno et al. 1998), using an expression plasmid for CapZ from Addgene (Plasmid #13451). To prepare B‐end capped F‐actin seeds, first mix 10 μM G‐actin and 0.1 μM CapZ (100:1) in G‐buffer (Section 2.2.1) and incubate the mixture for 10 min at room temperature. Then, add salts to the mixture (final concentration of 0.1 M KCl, 20 mM Hepes pH 7.4 and 2 mM MgCl₂) to start polymerization, and polymerize with CapZ overnight on ice. The prepared B‐end capped F‐actin seeds are diluted to a final concentration of 0.8 μM F‐actin and 8 nM CapZ in the bulk pointed‐end assembly assay described in the next section and as in our previous studies (Lewis et al. 2014; Yamashiro et al. 2014).

2.3.3. Pyrene‐Actin Polymerization Assay

F‐actin polymerization is monitored by measuring the fluorescence intensity of pyrene‐actin (excitation = 366.5; emission = 407 nm, Figure 1B), using a fluorometer with a cuvette (four sides clear) or a microplate fluorometer. Regarding the cuvette materials, quartz has the highest transmittance, but plastic cuvettes can also be utilized. Since actin polymerization is temperature‐sensitive, it is optimal to use a fluorometer with a temperature‐controlled system for the cuvettes, particularly if it is intended to measure final F‐actin levels at steady state (after 18–24 h) (Zimmerle and Frieden 1986). A water‐jacketed cuvette holder is useful but is not necessary for measuring pyrene‐actin fluorescence at room temperature.

2.3.4. Required Materials

• B‐end capped F‐actin seeds prepared as above.

• 10 μM 8% pyrene G‐actin: Mix pyrene‐G‐actin and unlabeled G‐actin to make 8% pyrene‐G‐actin in 10 μM total G‐actin in G‐buffer (Section 2.2.1). Ultracentrifuge the 8% pyrene G‐actin solution at 100,000 × g for 1 h to remove aggregates and collect the supernatant.

  Note: Pyrene‐G‐actin is easily photobleached. Keep it in the dark and on ice.

• G‐buffer: 2 mM Tris (pH 8.0), 0.2 mM CaCl₂, 0.2 mM DTT, 0.2 mM ATP

• F‐buffer: 20 mM HEPES (pH 7.5), 0.1 M KCl, 2 mM MgCl₂, 1 mM DTT

• 10× Mg²⁺/EGTA buffer (1 mL): Mix 10 μL of 15 mM MgCl₂ (final 0.15 mM), 5 μL of 0.5 M EGTA (final 2.5 mM) and 985 μL ddH₂0. Make this buffer the same day as performing assays.

• 10× polymerization mixture (2 mL): 1 M KCl, 5 mM ATP, 20 mM MgCl₂, 10 mM EGTA.

  Note: All buffers should be filtered with 0.22 μm membranes before use and at room temperature.

2.3.4.1. Monitoring Actin Polymerization at Pointed Ends

1. Mix the required amount of B‐end capped F‐actin seeds with Tmod and Tpm in 1/5 of the final volume in a microtube. Adjust the volume with F‐buffer. Incubate the tube in a 25°C water bath.

2. Dilute 8% pyrene G‐actin with G‐buffer for a final concentration of 1.5 to 2.5 μM and add one‐tenth volume of 10 × Mg²⁺/EGTA buffer. The total volume of this mixture is adjusted to 7/10 of the final volume. Incubate the tube in a 25°C water bath for 3 min.

   Note: This step is performed to convert Ca ²⁺ ‐G‐actin monomers to Mg ²⁺ ‐G‐actin monomers, since Ca ²⁺ ‐actin and Mg ²⁺ ‐actin have different elongation rate constants. Without prior conversion to a homogenous species of Mg ²⁺ ‐actin before the initiation of elongation, measured rates would reflect the conversion rate together with the elongation rate.

3. Mix the B‐end capped F‐actin seed solution containing Tmod and/or Tpm (prepared in Step 1) with the solution of pyrene‐labeled and unlabeled G‐actin (prepared in Step 2), and then add 1/10 volume of 10 × polymerization mixture. Mix quickly with gentle vortexing and transfer the mixture to a fluorescence cuvette using a pipette tip with the tip cut off to avoid shearing the F‐actin seeds.

   Note: Perform this step as quickly as possible.

4. Place the cuvette in the fluorometer and measure fluorescence intensity over time. An example of kinetic measurements of pyrene‐actin polymerization with CapZ‐capped F‐actin seeds, with and without P‐end capping by Tmod in the presence of Tpm is shown in Figure 1B,C.

5. A flow chart of the experimental steps for an example experiment using a total volume of 300 μL is shown in Figure 2.

   Note 1: When comparing elongation rates under different conditions, perform all manipulations identically, such as incubation time and gentle vortexing in Step 3.

   Note 2: Pyrene photobleaches rapidly with excitation at 367 nm. A flash lamp in a plate reader generally avoids the problem. When using a fluorometer, using a shutter and spacing out the measurements is effective in reducing photobleaching. If neither is available, minimize the intensity of the exciting light (just enough for appropriate signal to noise emission). Confirm that photobleaching is minimal by measuring fluorescence emission for 30–60 min using the conditions employed in the assay.

FIGURE 2.

A flow chart of the experimental steps for a pyrene‐actin polymerization assay using a total volume of 300 μL.

2.3.5. Pyrene‐Actin Depolymerization Assay

In the bulk P‐end depolymerization assay, B‐end capped F‐actin (~10% pyrene‐actin) is diluted to below the critical concentration at the pointed end (~0.6 μM), and depolymerization from the pointed end is monitored (Weber et al. 1994; Yamashiro et al. 2008). Vitamin D‐binding protein (2 μM) (Weber et al. 1994) or Latrunculin A (1 μM) (Yamashiro et al. 2008) may be added when diluting B‐end capped F‐actin to increase the depolymerization rate and prevent re‐addition of actin subunits. However, a caveat is that, in addition to sequestering actin monomers, Latrunculin A may accelerate actin depolymerization by promoting phosphate dissociation of the terminal subunits at both F‐actin ends (Fujiwara et al. 2018).

2.3.5.1. Required Materials

• Buffers, G‐actin, and pyrene‐actin prepared as above.

• Vitamin D‐binding protein (Sigma‐Aldrich)

• Latrunculin A (Enzo Life Sciences)

2.3.5.2. Procedure

1. Prepare B‐end capped F‐actin by copolymerizing 10 μM G‐actin (10% pyrene‐actin) and 0.1 μM CapZ or gelsolin (100:1), using polymerization buffers as indicated above. Let actin polymerize overnight on ice.

2. Bring the B‐end capped F‐actin solution to room temperature. Mix Tmod and/or Tpm with B‐end capped F‐actin and incubate for approximately 20 min at room temperature.

3. Dilute the mixture to 0.5 μM F‐actin with F‐buffer in the presence of 2 μM vitamin D‐binding protein or 1 μM Latrunculin A, and transfer the solution to a cuvette. Monitor changes in pyrene fluorescence using a spectrofluorometer.

3. Single Filament TIRFM Assays for P‐End Dynamics

3.1. Advantages of Single Filament Approaches

Bulk pyrene assays have traditionally been the technique of choice to investigate the effects of regulatory proteins on actin dynamics (Kouyama and Mihashi 1981). Bulk approaches, however, suffer from a major limitation due to the assumption that all reactions occurring in the sample are identical. As a result, bulk approaches fail to distinguish heterogeneities in reactions (Shekhar and Carlier 2016). Additionally, since pyrene assays only measure the amount of F‐actin in the sample, it is not possible to distinguish nucleation from accelerated elongation. The advent of total internal reflection fluorescence microscopy (TIRF) helped overcome some of these limitations. TIRF enabled direct real‐time observation of actin dynamics at the scale of individual filaments (Amann and Pollard 2001; Fujiwara et al. 2002). Single‐molecule imaging by TIRF also allows direct visualization of individual fluorescently labelled molecules of actin binding proteins directly interacting with actin filaments, providing spatial insights inaccessible via bulk approaches. TIRF has been especially helpful in studying reactions that lead to large changes in elongation/depolymerization at filament barbed ends (Paul and Pollard 2009; Shekhar et al. 2015; Ulrichs et al. 2023), Arp2/3 complex‐mediated branching (Smith et al. 2013), filament severing by cofilin (Andrianantoandro and Pollard 2006) as well as measurement of polymerization kinetics at the pointed ends of actin filaments (Fujiwara et al. 2007; Kuhn and Pollard 2005). Nevertheless, biochemical interactions that only cause a small change in the elongation/depolymerization rate, such as those occurring at filament pointed ends, have been much more difficult to study using conventional TIRF. Actin dynamics at the pointed end are more than one order of magnitude slower; for example, bare actin filaments depolymerize at 0.2 subunits/s from their pointed ends in comparison to ~10 subunits/s at the barbed end (Pollard 1986). Furthermore, the critical concentration for pointed ends is about 6‐fold higher than for barbed ends. As a result, a much higher actin monomer concentration is needed to observe noticeable pointed‐end elongation. Higher actin concentration, however, results in a higher background fluorescence signal as well as enhanced nucleation, which makes it very difficult to monitor the dynamics of individual filaments.

Recent implementation of microfluidics‐assisted TIRF (mf‐TIRF) microscopy has helped tackle many of these challenges (Figure 3) (Shekhar and Carlier 2016). In mf‐TIRF, actin filaments are attached to the glass coverslip through one of their ends. Mf‐TIRF facilitates simultaneous high‐throughput recording of 100s of actin filaments. Filament alignment along the microfluidic flow reduces the Brownian fluctuations of filament ends, which allows identification of small changes in growth/depolymerization rates of filament pointed ends. As a result, mf‐TIRF has helped resolve long‐standing questions relating to pointed‐end depolymerization by cofilin (alone and together with Cyclase‐associated protein) as well as processive pointed‐end elongation by bacterial effector protein VopF (Kotila et al. 2019; Kudryashova et al. 2022; Shekhar and Carlier 2017; Shekhar et al. 2019; Towsif et al. 2024; Towsif and Shekhar 2023; Wioland et al. 2017). This approach will be optimal to explore the precise mechanisms underlying Tpm enhancement of Tmod pointed end capping activities, as well as the proposed functions of the larger Tmod‐family members, Lmods, in promoting elongation at pointed ends and antagonizing the capping function of Tmods (Tolkatchev et al. 2020; Weber et al. 1994).

FIGURE 3.

(A) Schematic representation of the PDMS chamber used for microfluidics‐assisted TIRF (mf‐TIRF) imaging of individual actin filaments. (B) Left: A representative field of view containing actin filaments anchored at their barbed ends on the glass coverslip. The distal pointed ends are free, and the filaments are aligned under the flow. Right: The methodology used for determining the time‐point of arrest of P‐end growth by Tmod from kymographs. Scale bar, 10 μm. (C) Schematic representation of the experimental strategy used for investigating pointed‐end capping by Tmod. A solution containing G‐Actin was introduced into a mf‐TIRF flowcell in which Biotinylated SNAP‐CapZ was anchored on the glass coverslip. Pre‐formed filaments, captured by Biotinylated‐CapZ at their barbed ends, continued to elongate at their pointed ends by polymerization of G‐Actin in the solution. A solution containing G‐Actin (10 μM) and Tmod1 (0.5 μM) (in TIRF buffer) was then flowed into the chamber, and filament elongation was monitored until each filament paused. (D) Representative kymograph showing an Alexa‐488 labelled actin filament bound to coverslip‐anchored CP at its barbed end and elongating from its pointed end in the presence of Alexa‐488 G‐Actin (green bar) and Tmod1 (gray bar). White arrowhead denotes capping of the filament pointed end by Tmod1 (white arrow). BE, barbed end; PE, pointed end.

3.2. Preparation of Fluorescent‐Actin, Coverslips, and Imaging Chambers

3.2.1. Fluorescence Labelling of G‐Actin for mf‐TIRF Imaging

3.2.1.1. Required Materials

• Purified G‐actin (Section 2.2.1)

• G‐buffer (5 mM Tris–HCl pH 7.5, 0.5 mM Dithiothreitol (DTT), 0.2 mM ATP and 0.1 mM CaCl₂). Titrate pH at 4°C.

• Modified‐F‐buffer (20 mM PIPES pH 6.9, 0.2 mM CaCl₂, 0.2 mM ATP and 100 mM KCl). Titrate pH at 4°C.

• Alexa—488 Succinimidyl ester dye (Thermo Fisher Scientific, A20000)

• 5 mL Dounce homogenizer (Wheaton, 358,034)

• Ultra‐centrifuge

3.2.1.2. Procedure

1. Polymerize 2 mL of ~50 μM unlabeled G‐actin overnight by dialyzing against modified F‐buffer.

2. Next morning, add a five‐fold molar excess of Alexa‐488 dye to the F‐actin solution. Mix by vortexing.

3. Incubate for 2 h at room temperature with slow rotational mixing.

4. Pellet F‐actin by centrifugation at 450,000 × g in a table‐top ultracentrifuge (Beckman Optima MAX) for 40 min at room temperature.

5. Discard supernatant containing the free dye. Resuspend the pellet in 2 mL G‐buffer using a Dounce homogenizer.

6. Transfer the F‐actin solution to a microcentrifuge tube and incubate the tube on ice for 2 h to let the F‐actin depolymerize.

7. Add KCl (100 mM final concentration) and MgCl₂ (1 mM final concentration) mix to polymerize monomers into filaments. Incubate 1 h on ice.

8. Pellet F‐actin by centrifugation for 40 min at 450,000 × g at 4°C.

9. Discard the supernatant, which contains unpolymerized actin and free dye.

10. Add 2 mL of G‐buffer to the pellet and homogenize the pellet in a Dounce homogenizer. It can be helpful to let the pellet sit in the G‐buffer on ice for 20–30 min to make it easier to homogenize.

11. Dialyze overnight at 4°C against 1 L of G‐buffer to depolymerize labeled actin filaments into monomers.

12. Next morning, pre‐clear the solution by centrifugation at 450,000 × g for 40 min at 4°C to remove residual polymerized actin filaments.

13. Discard the pellet and collect the supernatant, which contains fluorescently labelled G‐actin.

14. Determine the concentration and labeling efficiency of actin by measuring the absorbance at 280 nm (A₂₈₀) and at 495 nM (A₄₉₅). The molar extinction coefficient for actin, ${\mathrm{\epsilon }}_{\mathrm{A}280}$= 45,840 M⁻¹ cm⁻¹ and for Alexa‐488 dye, ${\mathrm{\epsilon }}_{\mathrm{D}280}$ = 71,000 M⁻¹ cm⁻¹. The correction factor for the Alexa‐488 fluorophore at 280 nm is CF₂₈₀ = 0.11.

15. Calculate the concentration of actin and dye as follows:

    $$\left[\text{Actin}\right]=\frac{\left(A_{280}-A_{495}*{\mathit{CF}}_{280}\right)}{{\epsilon }_{P280}}$$

    $$\left[\mathrm{Dye}\right]=\frac{\left(A_{495}\right)}{{\epsilon }_{D280}}$$

    $$\%\text{labelling}=\frac{\left[\mathrm{Dye}\right]}{\left[\text{Actin}\right]}*100$$

16. The same strategy can also be used for labeling G‐actin with any other succinimidyl ester‐functionalized dyes.

3.2.2. Cleaning Glass Coverslips for Microfluidic (mf)‐TIRF Imaging

3.2.2.1. Required Materials

• Glass coverslips (#1.5, Fisher Scientific)

• Multi‐slide holder

• Water bath sonicator

• 1 M Potassium Hydroxide Solution

• 1 M HCl solution

• Ethanol 200 proof

• Deionized distilled water

• Liquid dishwasher detergent (any generic supermarket detergent).

3.2.2.2. Procedure

1. Place coverslips sideways in the multi‐coverslip holder. Ensure that the coverslips are not touching each other.

2. Fill the holder with deionized water, add two drops of dishwasher detergent, and place the chamber in a water bath sonicator. Sonicate for 20 min.

3. Decant the detergent solution. Rinse twice thoroughly with deionized water.

4. Fill the holder with 1 M KOH solution and sonicate for 20 min in a water bath sonicator.

5. Decant the KOH solution. Rinse twice thoroughly with deionized water.

6. Fill the holder with 1 M HCl solution and sonicate for 20 min in a water bath sonicator. Note that KOH and HCl help remove inorganic contaminants bound to the coverslips.

7. Decant the HCl solution. Rinse twice thoroughly with deionized water.

8. Fill the holder with 200 proof ethanol and sonicate for 20 min in a water bath sonicator. Note, ethanol helps remove all the organic contaminants bound to the coverslips.

9. Decant and replace with fresh ethanol.

10. The clean coverslips can now be stored in ethanol for up to a month. Prior to TIRF experiments, the coverslips are removed from ethanol and dried under a stream of compressed nitrogen.

3.2.3. Preparation of Polydimethylsiloxane (PDMS) Chambers

3.2.3.1. Required Materials

• Sylgard 184 Silicone Elastomer Kit (Dow Inc.)

• Molds for PDMS casting to make a three‐inlet, one‐outlet PDMS chamber with the following channel dimensions—40 μm in height, 1.5 cm in length, 800 μm in width (Shekhar 2017).

• Biopsy punch (Electron Microscopy Sciences, 69,039–07)

• Glass beaker

• Oven (70°C)

• Vacuum desiccator

• Surgical scalpel

3.2.3.2. Procedure

1. Add 50 g of the PDMS base to a glass beaker.

2. Add 5 g of curing agent to the beaker with PDMS base.

3. Mix vigorously using a spatula for at least 10 min.

4. Remove air bubbles created due to mixing by placing the beaker in a vacuum desiccator for about 1 h.

5. Place the mold in a petri dish. Pour the PDMS solution over the mold.

6. Place the petri dish in the oven and let the PDMS cure overnight.

7. Next morning, cut out PDMS microchambers with a scalpel.

8. Punch inlet and outlet holes in the PDMS chamber using a biopsy punch (Shekhar 2017).

3.3. Studying Capping of Filament Pointed Ends by Tmod (In Absence or Presence of Tpm)

3.3.1. Required Materials

• TIRF buffer (10 mM imidazole, pH 7.4, 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 0.2 mM ATP, 10 mM DTT, 1 mM DABCO (1,4‐diazabicyclo[2.2.2]octane))

• 10% Alexa 488‐labelled G‐actin

• Tmod protein (Section 2.2.2)

• Tpm protein (Section 2.2.3)

• PDMS chambers (Section 3.2.3)

• Precleaned microscopy coverslips (Section 3.2.2)

• Plasma cleaner (with oxygen cylinder)

• Bovine Serum Albumin, BSA (GoldBio, A‐420) dissolved in a 1% (by weight) solution in 20 mM HEPES pH 7.5 and 50 mM KCl.

• Streptavidin (Thermo Fisher Scientific, S888) dissolved in PBS to a final concentration of 30 mg/mL

• Maesflow microfluidic flow‐control system (Fluigent). A similar system can also be purchased from Elveflow, France.

• Biotinylated mouse capping protein (CP) (Shekhar et al. 2019)

• Poly L‐Lysine (PLL)‐PEG solution (1 mg/mL) containing biotinylated and unbiotinylated PLL‐PEG in a 1:40 ratio. (SuSos)

3.3.2. Procedure

1. Prepare pre‐formed actin filaments by adding 50 mM KCl, 1 mM MgCl₂, and 1 mM EGTA to 10% Alexa 488‐labelled G‐actin (10 μM final actin concentration, total). Incubate for 30 min at room temperature.

2. Plasma‐clean a precleaned coverslip in a plasma cleaner and attach it to a PDMS microchamber (~1 min in an oxygen‐saturated environment). Plasma cleaning aids in bonding between glass and the PDMS chamber.

3. Inject about 20 μL of PLL‐PEG solution into the microfluidic chamber using a pipette.

4. Incubate the PLL‐PEG solution (containing biotinylated and unbiotinylated PLL‐PEG in 1:40 ratio) for at least 15 min to allow passivation of the coverslip surface by PLL‐PEG.

5. Connect the microfluidics tubing of the Maesflow microfluidic flow‐control system. Rinse the chamber with TIRF buffer to remove unbound PLL‐PEG.

6. Introduce 4 μg/mL streptavidin and 1% BSA diluted in TIRF buffer and incubate in the chamber for 1 min at room temperature.

7. Rinse with TIRF buffer to remove unbound BSA and streptavidin.

8. Incubate the chamber with 1 nM biotinylated capping protein (CP) in TIRF buffer for 5 min.

9. Introduce 10% Alexa‐488 labelled G‐actin monomers under flow in TIRF buffer. Filaments nucleated in the solution will be captured by their barbed ends at surface‐anchored CP.

10. Allow attached filaments to grow at their pointed ends from G‐actin in the flow solution.

11. Replace flow solution with a solution containing 10% Alexa‐488 labelled G‐actin and Tmod in TIRF buffer.

12. Continue flowing the solution until all filaments in the field of view stop elongating (i.e., are capped by Tmod). Adjust the flow rate to ensure filaments remain in the evanescent field of the microscope.

13. Draw kymographs for all filaments and determine the time it takes for each individual filament to stop growing.

14. Plot the survival fraction of elongating filaments as a function of time (with t = 0 being the time at which Tmod was introduced into the chamber). Include at least 80–100 filaments in the analysis.

15. Fit the survival fraction plot (also called the Cumulative Distribution Function or CDF) with a single‐exponential function. The rate measured from the fit is referred to as the observed association rate constant k _obs .

16. Repeat the experiment for at least three different Tmod concentrations.

17. Plot the k_obs as a function of Tmod concentration.

18. Fit the curve to a linear fit. The slope of the fit gives the concentration‐dependent association rate constant of Tmod for the pointed end.

19. To determine the dwell time (or the off‐rate, k_off) of Tmod at pointed ends, rapidly cap all filament pointed ends with Tmod and then expose them to a solution containing G‐actin only (no Tmod).

20. Draw kymographs for all filaments and identify the time point when filaments resume elongation.

21. The average time it takes for filaments to resume elongation gives the dwell time of Tmod at the pointed end. The reciprocal of dwell time is the dissociation rate constant (k_off or off‐rate) of Tmod for pointed ends.

22. To study the effect of Tpm on Tmod's interaction with barbed ends—all reactions in steps 1 and 9–11 should be supplemented with 2 μM Tpm.

4. P‐End Assays in Living Cells

4.1. Advantages of Striated Muscle Cells

Isolated cardiac myocytes are ideal for deciphering mechanisms of actin pointed end dynamics and the acute requirements of actin binding proteins during myofibril assembly and development. Working with cultures of cardiac myocytes overcomes some of the limitations of animal models such as muscle thickness and challenges with uniform gene transfer. In culture, myocytes are large and flat with highly organized (polarized) thin (actin) filaments whose barbed and pointed ends are lined up in regular, repeating rows (striations) in sarcomeres. Moreover, the filament ends are ~1–2 μm apart so that pointed and barbed ends can be readily resolved by light microscopy, enabling analysis of actin dynamics at ends using fluorescent‐labeled actin introduced into cells (Figure 4) (Gokhin and Fowler 2017; Littlefield et al. 2001; Pappas et al. 2015). In addition, isolated cardiac myocytes are contractile so that physiological functions can be linked to subcellular structural changes. These cells are extremely amenable to functional manipulations including combinations of transductions and knockdowns (shRNA), as well as quantitative live cell microscopy. Consequently, the range of experimental conditions and approaches available with isolated myocytes provides useful insights into cellular and subcellular physiology. Isolated skeletal muscle myotubes are also suitable for studies of actin pointed end dynamics and function, although the larger sizes and thickness of myotubes as compared to cardiac myocytes make imaging of sarcomere thin filament ends more challenging (Pappas et al. 2010).

FIGURE 4.

Lmod2 Enhances Actin incorporation and dynamics at thin filament pointed ends. (A) Microinjection of Rho‐Actin in GFP (Upper) and GFP‐Lmod2 overexpressing (OE) (Lower) neonatal rat cardiomyocytes. Staining for α‐actinin marks the Z‐disk where the barbed ends of the Actin filament are located (pink arrows); pointed ends are denoted by blue arrowheads. GFP‐Lmod2 often localizes to the pointed end and along the length of the thin filament but is excluded from the Z‐disk; the non–pointed‐end localization is likely of low affinity and/or nonspecific (Tsukada et al. 2010) (Scale bar: 1 μm.) (A′) Plot profile of Rho‐Actin in cells transduced with GFP (pink) and GFP‐Lmod2 (orange). (B–D) FRAP of GFP‐cardiac Actin in rat cardiomyocytes transduced with mCherry or mCherry‐Lmod2. (B) Representative images of rat cardiomyocytes before and after photobleaching. Barbed (pink arrow) and pointed (blue arrowheads) ends of the Actin filaments. (Scale bar: 1 μm.) (C) Mean relative recovery following photobleaching over time ± SEM. (D) Mean slow and fast mobile fractions ± SEM; n = 9–15. *p < 0.05, ****p < 0.0001. Used with permission from (Pappas et al. 2015).

4.2. Assays of Actin Pointed‐End Dynamics With Fluorescence Recovery After Photobleaching

Fluorescence recovery after photobleaching (FRAP) is a microscopy‐based technique that allows the mobility rate of fluorescent molecules within a cell to be measured. Actin dynamics within cardiac thin filaments are measured by FRAP of fluorescently labeled actin; that is, labeled monomers (G‐actin) introduced into cells polymerize into filamentous actin (F‐actin) which are then bleached and subunit exchange is monitored by recovery of fluorescence intensity. Thin filaments are composed of polarized actin filaments, with both ends displaying actin turnover. The barbed ends are crosslinked within the Z‐disc and capped by CapZ, and the pointed ends extend toward the M‐line, where they are capped by tropomodulin (Tmod). Measurements of fluorescence recovery at the pointed and barbed ends of thin filaments plotted against time can determine the kinetics of actin polymerization at each end under various experimental conditions (e.g., expression of a signaling molecule of interest, treatment with various drugs, etc.). Actin dynamics at filament ends typically display the presence of two mobile fraction populations: a dynamic population with rapid fluorescence recovery (i.e., fast mobile fraction) that is dependent on muscle cell contractility, and a stable population (i.e., slow mobile fraction) that does not, or only slowly, recover fluorescence. The half‐time (t_1/2) of recovery to steady‐state fluorescence intensities is the time to recover 50% of the original fluorescence intensity and can be used to compare actin dynamics between thin filament barbed and pointed ends, and between treatment groups (Figure 4).

4.2.1. Required Materials

• Confocal microscope with environmental chamber preferably with 63x NA 1.4 objective (or better) and 488‐nm argon laser

• Matrigel GFR Membrane Matrix (Corning; CB‐40230A)

• 35 mm No. 1.5 coverslip glass‐bottomed dishes (MatTek; part number: P35G‐1.5‐20‐C)

• Dulbecco's Modified Eagle's Medium (DMEM) (Gibco; catalog # 11885084)

• Cardiomyocyte cell culture media: DMEM with the addition of 10% (vol/vol) FBS heat‐inactivated and 1% penicillin/streptomycin (Gibco; catalog #15140–122)

• Fluorescently tagged expression constructs: GFP‐cardiac actin (e.g., CellLight Actin‐GFP BacMam 2.0, ThermoFisher, catalog # C10582) plus potentially other proteins of interest tagged with mCherry (or similar fluorophore). Note that adenovirus expressing tagged protein constructs improves overall efficiency and levels of expression in the primary cardiomyocytes.

• Analysis software: Image J (https://imagej.net/ij/; NIH), Leica LASX, or similar

• Graphing software: GraphPad Prism (https://www.graphpad.com) or similar

4.2.2. Procedure

1. Prepare cardiomyocytes for FRAP experiment.

   1. Prepare Matrigel‐coating by diluting Matrigel Matrix with DMEM 1:1000.
   2. Coat 35 mm glass‐bottomed dishes with Matrigel coating by adding enough volume to cover the glass area. Let incubate for at least 30 min at 37°C at ∼5% CO₂ in a cell culture incubator before plating cells. Remove excess Matrigel coating and rinse the plate with sterile PBS before plating.
   3. Isolate primary cardiomyocytes from postnatal day 3 or younger mixed gender rats (e.g., Sprague–Dawley) or mice (e.g., C57BL/6J mice) by sequentially digesting excised hearts cut into small pieces in a collagenase/pancreatin solution. For a full cardiomyocyte isolation procedure see: (Brand et al. 2010). Plate cells at a range of densities starting with 150,000–250,000 cells/ml. Optimizing cell density is important for many reasons, including the health of the culture (cells mature better at higher densities), ease of FRAP at lower densities (single cells are easier to bleach) and effects on transfection/transduction efficiencies (Colpan et al. 2021; Ehler et al. 2013; Pappas et al. 2015; Sanger et al. 2010).
   4. Maintain cardiomyocytes at 37°C and ∼5% CO₂. Note that if necessary, to prevent fibroblast proliferation, add 1 μg/mL cytosine β‐d‐arabinofuranoside (Sigma, catalog # C1768) to the culture media 1 day after plating.
   5. Co‐transfect (or co‐transduce if using adenovirus vectors) cardiomyocytes with GFP‐cardiac actin and, depending on experimental design, a mCherry‐tagged protein of interest. For transfection, Lipofectamine 3000 (ThermoFisher, catalog # L3000008) or a similar reagent is utilized once the cardiomyocytes attach to the plate (~18 h after plating). For transduction with adenovirus, it is suggested to introduce 5–20 MOI of adenovirus 2–3 days after plating (Chu et al. 2018; Ehler et al. 2013).
2. FRAP experiment to determine actin dynamics. For more information, see review: (Lippincott‐Schwartz et al. 2018).

   1. Conduct FRAP experiment 5–6 days after plating.
   2. Acclimate the experimental dish with cardiomyocytes in the microscope environmental chamber set to 37°C and ∼5% CO₂.
   3. Select cardiomyocytes expressing GFP‐cardiac actin and mCherry‐protein of interest with relatively uniform fluorescence expression throughout the image area.
   4. Record 3–10 prebleach images of a single focal plane using a 63x objective from the selected cell. Make sure the image size captures an area larger than the photobleached area while allowing for rapid image capture.
   5. Photobleach a rectangle‐shaped area that includes 3–5 sarcomeres and make sure the area size is consistent throughout the experiment. Set the duration and laser power intensity to cause a reduction in intensity of around 50%–75% of the pre‐bleach value. The settings may differ depending on the confocal microscope. Avoid photobleaching the entire image area by adjusting acquisition times and adjusting to a lower laser power by 1%–5%. For example, using a Leica SP5‐II confocal microscope, photobleaching was accomplished with 80% total laser power for ~2 s.
   6. Monitor recovery after photobleaching by capturing images at successive intervals that reduce bleaching during acquisition, continuing until the intensity within the bleach region reaches a plateau. To accurately track the recovery process, capture 20–50 images at a high frequency (e.g., every 1 to 10 s depending on expected recovery rate) and confirm the recovery remains stable by taking 5 to 10 images at longer intervals (e.g., every min for 5–10 min). Interval times and durations may need to be adjusted depending on the confocal speed, image size, and recovery rates after photobleaching. For example, space imaging times and durations of 1 s (for a duration of 30 s), 5 s (for a duration of 150 s), and 10 s (for a duration of 600 s).
3. Analyze FRAP results.

   1. Import images into analysis software (e.g., Leica LAS AF Lite software version 4.0 or ZEN microscopy software version 2.5).
   2. Independently measure the fluorescence recovery at the pointed and barbed ends of thin filaments. Determine recovery independently from at least three sets of barbed and pointed thin filament ends per cell (e.g., barbed ends aligned at Z lines and pointed ends aligned at H zones for three sarcomeres), from 6 to 10 cardiomyocytes from three independent cultures.
   3. Plot the measurements of fluorescence recovery at the pointed and barbed ends against time after photobleaching using graphing software (e.g., GraphPad Prism version 10.0.0 or newer).
   4. Fit the data using a nonlinear regression two‐phase association curve using the two‐exponential association equation:

$$R=M_{\text{fast}}\times \left[1-\exp \left(-k_{\text{fast}}\times t\right)\right]+M_{\text{slow}}\times \left[1-\exp \left(-k_{\text{slow}}\times t\right)\right]$$

R = relative recovery at time t.

M = mobile fraction.

k = rate constant.

Nonlinear regression analysis provides quantitative information about kinetics through flexible modeling of the recovery data even under non‐ideal behaviors (e.g., slow diffusion, incomplete plateau, or other experimental factors). If fitting the two‐phase exponential equation to the fluorescence recovery data does not provide an R ² value > 0.70 or does not converge, the calculated parameters are deemed unreliable and should not be included in the analysis.

• eFrom the nonlinear regression curves, compare the t _1/2 values between experimental groups to determine significance. The slow versus fast mobile fractions at the barbed and pointed ends for the various experimental conditions can be compared by plotting a bar graph of the mean mobile fractions. For example, see (Colpan et al. 2021).
• fPerform statistical analyses using GraphPad Prism (or similar software). For comparisons of multiple groups, use a two‐way ANOVA with Tukey's post hoc test with a confidence interval level of 95%; differences with p < 0.05 are considered statistically significant. Note that each measurement from a single cell can be defined as a replicate since the cells are derived from 10 to 15 neonatal rat pups per culture.

4.3. Measurement of Fluorescent‐Actin Incorporation at Thin Filament Pointed‐Ends

Actin incorporation into the thin filament can be visualized via microinjecting fluorescently labeled (e.g., Rhodamine‐labelled) G‐actin into cardiomyocytes. After a relatively short incubation (up to 1 h) to allow G‐actin incorporation only at the ends of the thin filament, cardiomyocytes are fixed and stained with fluorescent phalloidin to label the entire thin filament length, or immunostained for other sarcomeric components. Alternatively, cells and incorporation of labeled proteins can be visualized in real time. Analysis of fluorescence images determines the ratio of the intensity of Rhodamine‐labeled actin fluorescence at the barbed versus pointed ends of the thin filaments under various experimental conditions. By use of this method, one can determine the mechanism by which various actin‐binding and/or regulatory proteins can promote or inhibit elongation of thin filaments (Littlefield et al. 2001; Pappas et al. 2015).

4.3.1. Required Materials

• Rhodamine‐labeled G‐actin (Cytoskeleton; catalog # AR05‐B)

• Resuspension buffer: 5 mM Tris (pH 8.0), 10 μM MgCl₂, 0.2 mM ATP, and 1 mM DTT

• Eppendorf microinjection system (old models 5246 and 5171or new InjectMan 4 and FemtoJet 4) plus injection capillary needles that are either purchased (Femtotips from Eppendorf, # 5242952008) or pulled for use.

• Inverted microscope equipped with phase contrast optics and long working distance 10x, 20x, and 40x dry objectives

• Relaxing buffer: 150 mM KCl, 5 mM MgCl₂, 10 mM 3‐(N‐morpholino) propanesulfonic acid (MOPS; pH 7.4), 1 mM EGTA, and 4 mM ATP (added fresh)

• Fixation buffer: 2% formaldehyde (Electron Microscopy Sciences; catalog #15710) prepared in relaxing buffer

4.3.2. Procedure

1. Microinject cardiomyocytes.

   1. Culture cardiomyocytes plated on coated glass coverslips in desired experimental conditions (e.g., drug treatment) for 4–7 days at 37 °C and ∼5% CO₂. For ease of identifying injected cells later in the experiment, cultured cells are often plated on gridded coverslips (MatTek 1.5, 18 mm, Fisher Scientific, catalog # NC1843909) or on coverslips that have been marked with a diamond pen to indicate quadrants.
   2. Resuspend Rhodamine‐labeled G‐actin to 1 mg/mL (20–100 μL depending on amount resuspended for the experiment) in resuspension buffer. Note: centrifuge the suspension at 100,000 x g for 30 min at 4°C to remove any filamentous actin that may have formed and/or particles that form over time and could clog the needle.
   3. Prior to microinjection, add fresh medium supplemented with 20 mM HEPES, pH 7.4.
   4. Using an inverted microscope equipped with a microinjection system, introduce the prepared Rhodamine‐labeled G‐actin into beating cardiomyocytes. To minimize injury to cells during microinjection, beating can be suspended via pre‐treating cells for 1 h with culture media containing 15 mM HEPES pH 7.4 and 20 mM 2,3‐butanedione monoxime (BDM, Sigma, catalog #B0753‐25G) (Gregorio et al. 1995).
   5. Incubate cells for various times up to 1 h at 37°C and ∼5% CO₂ to allow actin incorporation preferentially at the thin filament ends (Littlefield et al. 2001).
   6. Note, incubation for many hours or overnight is required for thin filament turnover and incorporation of actin all along the filament length (Skwarek‐Maruszewska et al. 2009; Wang et al. 2005).
2. Analyze actin incorporation into cardiomyocytes.

   1. After microinjecting cardiomyocytes, remove media, wash cells twice with PBS, and then incubate cells in relaxing buffer for 15 min at room temperature.
   2. Fix relaxed cardiomyocytes with fixation buffer for 15 min at room temperature.
   3. Stain coverslip with antibodies to striated muscle α‐actinin (clone EA‐53, Sigma, A7811) to identify Z lines and/or a green fluorescent‐phalloidin (e.g., Alexa‐488 phalloidin) to determine the localization of injected proteins with respect to thin filament polarity, and to ascertain myofibril maturation in cardiomyocytes.
   4. Image microinjected cardiomyocytes expressing Rhodamine‐labeled G‐actin.
   5. Using image analysis software (e.g., NIH Image J or DDecon; see below), perform line scans and determine the ratio of the intensity of Rho‐actin fluorescence at the pointed vs. barbed ends of thin filaments (see below, next section).

4.4. Thin Filament Length Measurements

Precisely regulated thin filament lengths are essential for efficient contractile force and proper heart function. Changes in the expression of actin regulatory proteins (e.g., Lmods, Tmods, nebulin, etc.), or changes in sarcomere length, can alter actin dynamics at filament pointed ends and control thin filament lengths (e.g., (Ahrens‐Nicklas et al. 2019; Gokhin and Fowler 2013; Kolb et al. 2016; Mi‐Mi et al. 2020; Ono 2010; Winter et al. 2016)). Intensity line scans of fluorescently stained actin filaments in cardiomyocytes are used to measure thin filament lengths under various experimental conditions. The utilization of curve fitting software to determine the full width half maximum (FWHM) from the line scans increases overall consistency between measurements and can provide up to ~50 nm precision in measurements of thin filament lengths (Littlefield and Fowler 2002; Schultz et al. 2023).

4.4.1. Required Materials

• Phosphate Buffered Saline pH 7.2 (PBS)

• Relaxing buffer: 10 mM MOPS, pH 7.4, 150 mM KCl, 5 mM MgCl₂, 1 mM EGTA, and 4 mM ATP (add ATP fresh)

• Fixation buffer: 2% formaldehyde (Electron Microscopy Sciences; catalog #15710) prepared in relaxing buffer

• Fluorescent‐phalloidin (e.g., Texas Red‐X Phalloidin from Life Technologies; catalog #T7471) or anti‐cardiac actin antibody (American Research Products; catalog # 03–61,075) followed by Alexa Fluor 488‐conjugated goat anti‐mouse IgG (ThermoFisher; catalog #A32723)

• Curve Fitting Software: DDecon plugin (https://bio.tools/ddecon) for ImageJ (https://imagej.net/ij/; NIH) or Fityk software (https://fityk.nieto.pl/)

• Microsoft Excel Spreadsheet software

4.4.2. Procedure

1. Obtain cardiomyocyte images.

   1. Culture cardiomyocytes are plated on coated glass coverslips in desired experimental conditions.
   2. Prior to fixation, 4–7 days after plating, remove media, wash cells twice with PBS, and then incubate cells in relaxing buffer for 15 min at room temperature.
   3. Fix relaxed cardiomyocytes with fixation buffer for 15 min at room temperature.
   4. Stain the coverslip with fluorescent‐conjugated phalloidin or anti‐cardiac‐actin antibody followed by a fluorescent‐tagged secondary antibody to fluorescently stain actin‐containing thin filaments. Additional antibodies such as those specific for α‐actinin can be used to identify the structural maturity of the myofibrils in the cardiomyocytes. Anti‐Tmod1 antibodies can also be used to mark the pointed ends of the thin filaments.
   5. Image cardiomyocytes containing myofibrils displaying uniform actin staining along thin filaments, using 63x or 100x objective. For accurate analysis, image a minimum of 10 cardiomyocytes per experimental condition from 3 separate cultures. For best results, if using a widefield microscope, deconvolve a Z‐stack using an inverse matrix algorithm or blind deconvolution if the point spread function is not well characterized. Select an image of an optical plane containing actin staining that is most in focus.
2. Measure thin filament lengths using Distributed Deconvolution (DDecon) software (Littlefield and Fowler 2002). For methodological details, see (Gokhin and Fowler 2017). For an alternative, use Fityk curve fitting software to obtain thin filament length measurements (Kiss et al. 2020).

   1. Import images into the DDecon subfolder on the computer. Note that DDecon has not been updated. As such, it only runs on computers with ImageJ Version 1.52 DDecon plug‐in.
   2. Launch DDecon plugin in ImageJ and add experiment to link measurements into a single file.
   3. Load actin filament images into the program.
   4. Select image and create a line scan from pointed end to pointed end across the Z line (i.e., gap to gap in actin filament staining pattern) spanning a minimum of one thin filament array. For consistent results, try to measure 2–10 adjacent thin filament arrays with each line scan along a myofibril.
   5. Click ‘Add Line Scan’ in the LSD Decon Launch screen after each line scan drawn.
   6. Once all line scans for one image are collected, click ‘Line Scan Analysis’ and refine the individual line scans if needed in the Line Scan screen.
   7. Click ‘Do DDecon’ to perform model fitting and to obtain length measurement. Adjust peak profiles with model profile.
   8. Click ‘Fit Profile’ and make sure that under ‘Results Summary’ the error is < 10%; otherwise, discard the line scan.
   9. Save results in a spreadsheet.
3. Analyze thin filament lengths.

   1. Open spreadsheet with line scan measurements.
   2. The thin filament length is found in the column titled ‘Length’. This value is the average of the full width half maximum of the modeled peaks corresponding to the thin filament lengths in each line scan. It is important to note that this measurement is given in pixels. Calculate the pixels/μm measurement from the microscope to convert the values to μm.
   3. The sarcomere length is found in the column titled ‘Average Z‐Z Distance’. This value is the average distance between each peak. Again, these values are given in pixels and need to be converted to μm.
   4. To determine significant changes in thin filament length, compare the thin filament lengths between experimental conditions. Perform statistical analyses using Prism (or similar software). For comparisons of multiple groups, use a two‐way ANOVA with Tukey's post hoc test with a confidence interval level of 95%; differences with p < 0.05 are considered statistically significant. Note that each measurement from a single cell should be defined as a replicate, since the cells are derived from 10 to 15 neonatal rat pups per culture.

Conflicts of Interest

The authors declare no conflicts of interest.

Yamashiro, S. , Shekhar S., Novak S. M., Biswas S., Gregorio C. C., and Fowler V. M.. 2025. “Actin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin.” Cytoskeleton 82, no. 9: 571–591. 10.1002/cm.22007.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.