Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: protein crosslinking studies

When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.     

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca²⁺-dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca²⁺ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca²⁺ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca²⁺ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca²⁺-dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca²⁺-dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.     

Plant ERD2s self-interact and interact with GTPase-activating proteins and ADP-ribosylation factor 1

ERD2s (ER luminal protein receptors)-mediated retrograde transport is one of the most substantial processes to maintain the endoplasmic reticulum (ER) homeostasis. It is completed by the recognition of the escaped ER luminal proteins, the gathering into COP I vesicle, and the fusion and releasing into the ER. ERD2s can recognize HDEL/KDEL motifs at the C-terminal of the escaped ER luminal proteins at the Golgi to initiate the retrograde transport. However, these mechanisms remain largely unknown in plants. We recently found that two Nicotiana benthamiana homologs, ERD2a and ERD2b, functioned as ER luminal protein receptors, were required for both HDEL/KDEL motifs-mediated ER retrieval and participated in cell death triggered by ER stress and nonhost pathogens. Here, we provide a set of new data that ERD2a/2b can form homo- or hetero-oligomerization and interact with both the ADP-ribosylation factor 1 (ARF1) and its potential GTPase-activating proteins (GAP) indicated by the firefly luciferase complementation imaging assay (LCI). These evidences further support the ER luminal protein receptor function of ERD2a/2b in plants and suggest their evolutionarily conserved mechanism during the retrograde trafficking. We also analyze the characteristics of ERD2s within a species and among different species.     

Cytochemical localization of membrane-bound Mg2+-dependent ATPase activity in guinea pig sperm head before and during the acrosome reaction

The distribution of ATPase activity in the heads of uncapacitated, capacitated, and acrosome-reacting guinea-pig spermatozoa was examined cytochemically using the Wachstein-Meisel's technique. In uncapacitated spermatozoa, the reaction products of the enzyme activity were localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity was Mg²⁺-dependent and inhibited by both Ca²⁺ and SH-blocking agents. This Mg²⁺-dependent ATPase activity was also demonstrated at the same sites in capacitated spermatozoa, whereas it was completely absent in acrosome-reacting spermatozoa. Although we did not determine the exact time of inactivation of the enzyme, it appeared to occur before the plasma membrane fused with the underlying outer acrosomal membrane. The abrupt loss of the Mg²⁺-dependent ATPase activity in the plasma and outer acrosomal membranes immediately before the onset of the acrosome reaction seems to suggest that inactivation of this enzyme by Ca²⁺ is one of the important biochemical events involved in the acrosome reaction.     

GnRH-Bik/Bax/Bak chimeric proteins target and kill adenocarcinoma cells; the general use of pro-apoptotic proteins of the Bcl-2 family as novel killing components of targeting chimeric proteins

In recent years chimeric proteins carrying bacterial toxins as their killing moiety, have been developed to selectively recognize and kill cell populations expressing speciific receptors. The involvement of Gonadotropin releasing hormone (GnRH) has been demonstrated in several adenocarcinomas and a GnRH-bacterial toxin chimeric protein (GnRH-PE66) was thus developed and found to specifically target and kill adenocarcinoma cells both in vitro and in vivo. Because of the immunogenicity and the non-specific toxicity of the bacterial toxins, we have developed new chimeric proteins, introducing apoptosis inducing proteins of the Bcl-2 family as novel killing components. Sequences encoding the human Bik, Bak or Bax proteins were fused to the GnRH coding sequence at the DNA level and were expressed in E. coli. GnRH-Bik, GnRH-Bak and GnRH-Bax new chimeric proteins efficiently and specifically inhibited the cell growth of adenocarcinoma cell lines and eventually led to cell death. All three Bcl2-proteins-based chimeric proteins seem to induce apoptosis within the target cells, without any additional cell death stimulus. Apoptosis-inducing-proteins of the Bcl-2 family targeted by the GnRH are novel potential therapeutic reagents for adenocarcinoma treatment in humans. This novel approach could be widely applied, using any molecule that binds a specific cell type, fused to an apoptosis-inducing protein.     

Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin, DcKRP120-2

We have previously described the biochemical isolation of 65 kDa and 120 kDa microtubule-associated proteins from carrot cytoskeletons. The 65 kDa MAPs have subsequently been shown to be structural MAPs that reconstitute 30 nm cross-bridges of the kind that maintain cortical microtubules in parallel groups. By exploiting its avid binding to microtubules, we have now devised a method for isolating MAP120 from protoplast extracts, and shown that it has properties of a kinesin-related protein. MAP120 segregates with the cold stable pool of microtubules in carrot cytoskeletons, whilst the 65 kDa MAPs are also associated with the cold-sensitive microtubules. On gradient gels, MAP120 resolves as two kinesin-like bands. We report the isolation of a carrot cDNA, DcKRP120-2, corresponding to a novel kinesin of the BimC class known to move to the plus ends of microtubules. Antibodies raised against specific expressed sequences recognize the upper band, while the lower band is recognized by antibodies to the tobacco kinesin-related protein, TKRP125. We have also isolated a partial genomic carrot DNA, DcKRP120-1, homologous to the motor region of tobacco TKRP125. Immunofluorescence of the two proteins produces different staining patterns. Anti-TKRP125 labels the cortical microtubules and the pre-prophase band, but anti-DcKRP120-2 does so only weakly. Both clearly stain the spindle and the phragmoplast, but in a proportion of cells anti-DcKRP120-2 strongly decorates the phragmoplast mid-line where the plus ends of the microtubules overlap. We discuss the potential roles of these proteins during the microtubule cycle.     

RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules

In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)(+) RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R(+) SGs is initiated by the phosphorylation of eIF-2alpha. A phosphomimetic eIF-2alpha mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2alpha mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2alpha to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.     

Detection of extracellular vesicles in the mouse vaginal fluid: Their delivery of sperm proteins that stimulate capacitation and modulate fertility

Extracellular vesicles (EVs) were isolated by ultracentrifugation of vaginal luminal fluid (VLF) from superovulated mice and identified for the first time using transmission electron microscopy. Characterized by size and biochemical markers (CD9 and HSC70), EVs were shown to be both microvesicular and exosomal and were dubbed as “Vaginosomes” (VGS). Vaginal cross-sections were analyzed to visualize EVs in situ: EVs were present in the lumen and also embedded between squamous epithelial and keratinized cells, consistent with their endogenous origin. Western blots detected Plasma membrane Ca²⁺-ATPase 1 (PMCA1) and tyrosine-phosphorylated proteins in the VGS cargo and also in uterosomes. Flow cytometry revealed that following coincubation of caudal sperm and VLF for 30 min, the frequencies of cells with the highest Sperm adhesion molecule 1 (SPAM1), PMCA1/4, and PMCA1 levels increased 16.4-, 8.2-, and 27-fold, respectively; compared with control coincubated in phosphate buffered saline (PBS). Under identical conditions, sperm tyrosine-phosphorylated proteins were elevated ~3.3-fold, after VLF coincubation. Progesterone-induced acrosome reaction (AR) rates were significantly (p < 0.001) elevated in sperm coincubated with VGS for 10–30 min, compared with PBS. Sperm artificially deposited in the vaginas of superovulated females for these periods also showed significant (p < 0.01) increases in AR rates, compared with PBS. Thus in vitro and in vivo, sperm acquire from the vaginal environment factors that induce capacitation, explaining recent findings for their acrosomal status in the isthmus. Overall, VGS appear to deliver higher levels of proteins involved in preventing premature capacitation and AR than those promoting them. Our findings which have implications for humans open the possibility of new approaches to infertility treatment with exosome therapeutics.     

Characterization of the antigen recognized by a monoclonal antibody MN9: unique transport pathway to the equatorial segment of sperm head during spermiogenesis

Summary MN9, a monoclonal antibody raised against mouse spermatozoa, specifically recognizes the equatorial segment of sperm head in several mammalian species, including humans. Colloidal gold-immuno-electron microscopy of mouse spermatozoa has shown that the antigen is localized in the space between the outer and inner acrosome membranes and on the acrosome membranes at the equatorial segment. Immunoblotting after electrophoresis of spermatozoa from the cauda epididymidis has identified two immunoreactive bands: 38 kDa and 48 kDa in mouse, and 48 kDa in rat. During spermiogenesis in rat, this antigen is transported to the equatorial segment via a unique pathway, first appearing in some cisternae of the endoplasmic reticulum and in the Golgi apparatus of spermatids at around step 3. The antigen can further be found on the vesicles at thetrans-side of the Golgi apparatus, in the matrix of the head cap, and on the head cap membrane in step-4 to step-7 spermatids. The antigen appears to be concentrated at the equatorial segment during late spermiogenesis. Neither the (pro-)acrosomic granule nor the surrounding membrane are required in this pathway. This pathway can be termed the Golgi-head cap tract.     

Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause

Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis—life without water—during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H₂O₂) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.     

Identification and characterization of Cor413im proteins as novel components of the chloroplast inner envelope

Plastids are surrounded by two membrane layers, the outer and inner envelope membranes, which have various transport and metabolic activities. A number of envelope membrane proteins have been identified by biochemical approaches and have been assigned to specific functions. Despite those efforts, the chloroplast envelope membrane is expected to contain a number of as yet unidentified proteins that may affect specific aspects of plant growth and development. In this report, we identify and characterize a novel class of inner envelope membrane proteins, designated as Cor413 chloroplast inner envelope membrane group (Cor413im). Both in vivo and in vitro studies indicate that Cor413im proteins are targeted to the chloroplast envelope. Biochemical analyses of Cor413im1 demonstrate that it is an integral membrane protein in the inner envelope of chloroplasts. Quantitative real-time PCR analysis reveals that COR413IM1 is more abundant than COR413IM2 in cold-acclimated Arabidopsis leaves. The analyses of T-DNA insertion mutants indicate that a single copy of COR413IM genes is sufficient to provide normal freezing tolerance to Arabidopsis. Based on these data, we propose that Cor413im proteins are novel components that are targeted to the chloroplast inner envelope in response to low temperature.     

Localization and quantitative expression of mRNAs encoding the testis-specific histone TH2B,the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat

Expression of the testis-specific histone TH2B, the phosphoprotein p19, and the transition proteins TP1 and TP2, was localized in the rat testis and quantified, using in situ hybridization of their mRNAs with radiolabeled probes and image analysis. In a first study, expression was assessed during testicular development between day 2 and day 65 postpartum. TH2B mRNAs appeared first in preleptotene spermatocytes (PL) on day 12 and in pachytene spermatocytes (PS) on day 18; p19 mRNAs were present in PS from day 18 onward, and TP1 and TP2 mRNAs were detected in round spermatids (RS) from day 32 onward. In the second trial, the expression of these four genes was studied throughout the cycle of spermatogenic epithelium in mature animals. TH2B mRNAs were localized in B spermatogonia at stage V, and in PL at stages VII and VIII but no longer in leptotene and zygotene spermatocytes. Thereafter, TH2B mRNAs were observed in PS from stages III–IV to XIII. The steady-state mRNA level per cell was high in PS with a maximum at stages IX–X. p19 mRNAs were present in PS from stages III–IV onward and in RS up to stages 1–2 of spermiogenesis. The maximum mRNA level per cell was observed in PS between stages VII and XIII. The presence of TP1 mRNAs was restricted to spermatids from steps 6 to 15–16 of spermiogenesis while TP2 mRNAs were detected in spermatids only between step 7 and step 13. The highest steady-state amounts of mRNAs were observed between step 7 and step 14 for TP1 and between step 10 and step 12 for TP2. Mol. Reprod. Dev. 51:22–35, 1998. © 1998 Wiley-Liss, Inc.     

Functional conservation of MBD proteins: MeCP2 and Drosophila MBD proteins alter sleep

Proteins containing a methyl‐CpG‐binding domain (MBD) bind 5mC and convert the methylation pattern information into appropriate functional cellular states. The correct readout of epigenetic marks is of particular importance in the nervous system where abnormal expression or compromised MBD protein function, can lead to disease and developmental disorders. Recent evidence indicates that the genome of Drosophila melanogaster is methylated and two MBD proteins, dMBD2/3 and dMBD‐R2, are present. Are Drosophila MBD proteins required for neuronal function, and as MBD‐containing proteins have diverged and evolved, does the MBD domain retain the molecular properties required for conserved cellular function across species? To address these questions, we expressed the human MBD‐containing protein, hMeCP2, in distinct amine neurons and quantified functional changes in sleep circuitry output using a high throughput assay in Drosophila. hMeCP2 expression resulted in phase‐specific sleep loss and sleep fragmentation with the hMeCP2‐mediated sleep deficits requiring an intact MBD domain. Reducing endogenous dMBD2/3 and dMBD‐R2 levels also generated sleep fragmentation, with an increase in sleep occurring upon dMBD‐R2 reduction. To examine if hMeCP2 and dMBD‐R2 are targeting common neuronal functions, we reduced dMBD‐R2 levels in combination with hMeCP2 expression and observed a complete rescue of sleep deficits. Furthermore, chromosomal binding experiments indicate MBD‐R2 and MeCP2 associate on shared genomic loci. Our results provide the first demonstration that Drosophila MBD‐containing family members are required for neuronal function and suggest that the MBD domain retains considerable functional conservation at the whole organism level across species.     

细菌脂多糖诱导人单核细胞株对细菌脂蛋白的耐受性及其与肌动蛋白骨架关系

细菌脂多糖(LPS)可诱导宿主对LPS的耐受,但对细菌脂蛋白(BLP)是否存在交叉耐受,目前报道不一。采用人单核细胞株(THP-1),建立小剂量LPS诱导THP-1对LPS耐受的细胞模型;观察细胞肌动蛋白骨架、炎症因子TNF-α、IL-1β、IL-6的浓度及NF-κB的DNA结合活力的变化情况;探讨BLP交叉耐受及细胞骨架在其中的作用。结果显示,THP-1细胞经小剂量(10ng/ml)LPS、大剂量(100ng/ml)LPS或BLP刺激后,细胞形态严重变形,肌动蛋白重组,细胞周边肌动蛋白丝带消失,出现明显的肌动蛋白收缩团块及伪足,细胞核内NF-κB的DNA结合活性显著升高,培养上清液中炎症因子(TNF-α、IL-1β及IL-6)的释放显著增加;而小剂量LPS预刺激12h后,再用大剂量的LPS或BLP刺激6h,上述指标明显改善;采用细胞骨架肌动蛋白聚集破坏剂鬼笔环肽预处理后的THP-1细胞,可取消由小剂量LPS诱导的自身耐受及对BLP的交叉耐受;可见,细菌LPS、BLP(100ng/ml)可诱导THP-1细胞肌动蛋白骨架的改变,激活NF-κB信号通路,诱导炎性细胞因子TNF-α、IL-1、IL-6过度释放,激活宿主炎症细胞的炎症反应;而小剂量LPS预刺激后可诱导出THP-1细胞对LPS的自身耐受和对BLP的交叉耐受;细胞骨架肌动蛋白参与了小剂量LPS诱导THP-1细胞对LPS自身耐受和对BLP交叉耐受的形成。     

Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation. Published in Russian in Biokhimiya, 2008, Vol. 73, No.9, pp. 1274–1280.     

Histone H1 variants as sperm-specific nuclear proteins of Rana catesbeiana,and their role in maintaining a unique condensed state of sperm chromatin

Amino acid analyses of nuclear basic proteins of an anuran amphibian, Rana catesbeiana, revealed that they are comprised of a full set of core histones and three types of lysine-rich, sperm-specific proteins. On the basis of their amino-acid compositions and partial amino-acid sequences of their trypsin-resistant cores, the sperm-specific proteins could be defined as members of the histone H1 family. Both micrococcal nuclease digestion and electron microscopy indicated that sperm chromatin consists of nucleosomal and fibrillar DNA structures which are irregularly interspersed with each other. When sperm nuclei were incubated with nucleoplasmin, nuclei decondensed to some extent, and the sperm-specific H1s were removed, but not completely. The residual sperm-specific histone H1 variants were also found in reconstituted male pronuclear chromatin, comprising regularly spaced nucleosomes. We conclude that sperm-specific histone H1 variants are essential for chromatin condensation in the sperm nuclei, but that their complete removal is not necessary for the remodeling into somatic chromatin that takes place after fertilization. Mol. Reprod. Dev. 47:181–190, 1997. © 1997 Wiley-Liss, Inc.     

Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins

Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein.