Tektin makes a microtubule a "micropillar".

Inside sperm flagella, there are nine doublet microtubules composed of A and B tubules. In this issue of Cell, Leung et al. and Zhou et al. present high-resolution cryo-EM structures of doublet microtubules from mammalian sperms and show unprecedented structures of the A tubules, which are almost entirely occupied with tektin bundles.

Structural specializations of the sperm tail.

Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.

Structures of sperm flagellar doublet microtubules expand the genetic spectrum of male infertility.

Sperm motility is crucial for successful fertilization. Highly decorated doublet microtubules (DMTs) form the sperm tail skeleton, which propels the movement of spermatozoa. Using cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based modeling, we determined the structures of mouse and human sperm DMTs and built an atomic model of the 48-nm repeat of the mouse sperm DMT. Our analysis revealed 47 DMT-associated proteins, including 45 microtubule inner proteins (MIPs). We identified 10 sperm-specific MIPs, including seven classes of Tektin5 in the lumen of the A tubule and FAM166 family members that bind the intra-tubulin interfaces. Interestingly, the human sperm DMT lacks some MIPs compared with the mouse sperm DMT. We also discovered variants in 10 distinct MIPs associated with a subtype of asthenozoospermia characterized by impaired sperm motility without evident morphological abnormalities. Our study highlights the conservation and tissue/species specificity of DMTs and expands the genetic spectrum of male infertility.

Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility.

Spermatozoa must leave one organism, navigate long distances, and deliver their paternal DNA into a mature egg. For successful navigation and delivery, a sperm-specific calcium channel is activated in the mammalian flagellum. The genes encoding this channel (CatSpers) appear first in ancient uniflagellates, suggesting that sperm use adaptive strategies developed long ago for single-cell navigation. Here, using genetics, super-resolution fluorescence microscopy, and phosphoproteomics, we investigate the CatSper-dependent mechanisms underlying this flagellar switch. We find that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we find unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility.

Bi-allelic mutations in DNAH7 cause asthenozoospermia by impairing the integrality of axoneme structure.

Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of both the cilia and flagella, and defects in these proteins generally impair the axoneme structure and cause primary ciliary dyskinesia. But relatively little is known about the influence of dynein protein arm defects on sperm flagella function. Here, we recruited 85 infertile patients with idiopathic asthenozoospermia and identified bi-allelic mutations in DNAH7 (NM_018897.3) from three patients using whole-exome sequencing. These variants are rare, highly pathogenic, and very conserved. The spermatozoa from the patients with DNAH7 bi-allelic mutations showed specific losses in the inner dynein arms. The expression of DNAH7 in the spermatozoa from the DNAH7-defective patients was significantly decreased, but these patients were able to have their children via intra-cytoplasmic sperm injection treatment. Our study is the first to demonstrate that bi-allelic mutations in DNAH7 may impair the integrality of axoneme structure, affect sperm motility, and cause asthenozoospermia in humans. These findings may extend the spectrum of etiological genes and provide new clues for the diagnosis and treatment of patients with asthenozoospermia.

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure.

Conception in mammals is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum can be divided into the midpiece, the principal piece and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility; however, the actin organization in sperm remains elusive. In the present work, we show that there are different types of actin structures in the sperm tail by using three-dimensional stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.

Identification, Characterization and Synthesis of Walterospermin, a Sperm Motility Activator from the Egyptian Black Snake Walterinnesia aegyptia Venom.

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4.

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.

Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.

Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, ß-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.

Human sperm tail proteome suggests new endogenous metabolic pathways.

Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous pools, and thus to adapt to putative exogenous fluctuations.

Alternative splicing, promoter methylation, and functional SNPs of sperm flagella 2 gene in testis and mature spermatozoa of Holstein bulls.

The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella.

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.

SEPT12-microtubule complexes are required for sperm head and tail formation.

The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, mitosis, and vesicle trafficking by interacting with various cytoskeletons. Our previous studies have indicated that SEPTIN12+/+/+/- chimeras with a SEPTIN12 mutant allele were infertile. Spermatozoa from the vas deferens of chimeric mice indicated an abnormal sperm morphology, decreased sperm count, and immotile sperm. Mutations and genetic variants of SEPTIN12 in infertility cases also caused oligozoospermia and teratozoospermia. We suggest that a loss of SEPT12 affects the biological function of microtublin functions and causes spermiogenesis defects. In the cell model, SEPT12 interacts with α- and ß-tubulins by co-immunoprecipitation (co-IP). To determine the precise localization and interactions between SEPT12 and α- and ß-tubulins in vivo, we created SEPTIN12-transgene mice. We demonstrate how SEPT12 interacts and co-localizes with α- and ß-tubulins during spermiogenesis in these mice. By using shRNA, the loss of SEPT12 transcripts disrupts α- and ß-tubulin organization. In addition, losing or decreasing SEPT12 disturbs the morphogenesis of sperm heads and the elongation of sperm tails, the steps of which are coordinated and constructed by α- and ß-tubulins, in SEPTIN12+/+/+/- chimeras. In this study, we discovered that the SEPTIN12-microtubule complexes are critical for sperm formation during spermiogenesis.

A caged progesterone analog alters intracellular Ca2+ and flagellar bending in human sperm.

Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.

Proteomic profiling reveals compartment-specific, novel functions of ascidian sperm proteins.

In this study, we performed extensive proteomic analysis of sperm from the ascidian Ciona intestinalis. Sperm were fractionated into heads and flagella, followed by further separation into Triton X-100-soluble and -insoluble fractions. Proteins from each fraction and whole sperm were separated by isoelectric focusing using two different pH ranges, followed by SDS-PAGE at two different polyacrylamide concentrations. In total, 1,294 protein spots representing 304 non-redundant proteins were identified by mass spectrometry (MALDI-TOF). On comparison of the proteins in each fraction, we were able to identify the proteins specific to different sperm compartments. Further comparison with the testis proteome allowed the pairing of proteins with sperm-specific functions. Together with information on gene expression in developing embryos and adult tissues, these results provide insight into novel cellular and functional aspects of sperm proteins, such as distinct localization of actin isoforms, novel Ca(2+)-binding proteins in axonemes, localization of testis-specific serine/threonine kinase, and the presence of G-protein coupled signaling and ubiquitin pathway in sperm flagella.

Isolation of lipocalin-type protein from rainbow trout seminal plasma and its localisation in the reproductive system.

The lipocalin protein family is a large and diverse group of small extracellular proteins characterised by their ability to bind hydrophobic molecules. In the present study, we describe the isolation procedure for rainbow trout seminal plasma protein, characterised by a moderate migration rate during polyacrylamide gel electrophoresis, providing information regarding its basic features and immunohistochemical localisation. This protein was identified as a lipocalin-type protein (LTP). The molecular mass of LTP was found to be 18,848 Da and it was found to lack any carbohydrate components. Only a few Salmoniformes contain LTP in their seminal plasma. The abundance of LTP in the Sertoli and Leydig cells of the testes of the rainbow trout, as well as in secretory cells of the efferent duct, suggests that this protein is specific for rainbow trout milt, where it acts as a lipophilic carrier protein. Moreover, the specific localisation of LTP in the flagella of the spermatozoa suggests a role for LTP in sperm motility. Further experiments are necessary to identify the endogenous ligands for LTP in rainbow trout seminal plasma and to characterise the binding properties of this protein.

Mechanical properties of the passive sea urchin sperm flagellum.

In this study we used Triton X-100 extracted sea urchin spermatozoa to investigate the mechanical behavior of the basic 9+2 axoneme. The dynein motors were disabled by vanadate so that the flagellum is rendered a passive structure. We find that when a proximal portion of the flagellum is bent with a glass microprobe, the remainder of the flagellum distal to the probe exhibits a bend in the opposite direction (a counterbend). The counterbend can be understood from the prevailing sliding doublet model of axoneme mechanics, but does require the existence of elastic linkages between the outer doublets. Analysis of the shapes of counterbends provides a consensus value of 0.03-0.08/microm(2) for the ratio of the interdoublet shear resistance (E(S)) to the bending resistance (E(B)) and we find that the ratio E(S)/E(B) is relatively conserved for both passive flagella and transiently quiescent live flagella. This ratio expresses a fundamental mechanical property of the eukaryotic axoneme. It defines the contributions to total bending resistance derived from bending the microtubules and from stretching the interdoublet linkages, respectively. Using this ratio, and computer simulations of earlier experiments that measured the total stiffness of the flagellum, we obtain estimates of approximately 1 x 10(8) pN nm(2)/rad for E(B) and 6 pN/rad for E(S), assuming that both elasticities are linear. Our results indicate that the behavior of the flagellum is close to that predicted by a linear model for shear elasticity.

The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility.

Sub-fertility is a major problem in crossbred bulls. Identification of subtle differences in the quality of cryopreserved spermatozoa among bulls belonging to different fertility rankings would help determine the latent fertility of semen before their use at field conditions. In the present study, we assessed the status of tyrosine phosphorylation, membrane integrity and acrosome reaction of cryopreserved spermatozoa in crossbred bulls (n = 22) with different levels of field fertility and assessed their relationship with fertility. Bulls were categorized into above-average (n = 4), average (n = 14) and below-average (n = 4) based on their different field fertility rates. The progressive sperm motility was significantly (P < 0.05) higher in above-average fertile bulls compared to either average or below-average fertile bulls whereas sperm membrane integrity and acrosomal reaction status did not differ among the three groups. The proportion of live tyrosine-phosphorylated spermatozoa were significantly (P < 0.05) higher in below-average and average fertile bulls compared to above-average bulls. Immunolocalization of protein tyrosine phosphorylation in spermatozoa revealed that the proportion of spermatozoa showing tyrosine phosphorylation at acrosome and post-acrosomal area (APA) and at acrosome, post-acrosome and tail (APAT) were significantly (P < 0.05) higher in below-average fertile bulls than other groups. The APA pattern (r = -0.605; P < 0.01) and APAT (r = 0.507; P < 0.05) pattern were significantly and negatively correlated with bull fertility. It was concluded that the proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility.

Expression and localization of voltage-dependent anion channels (VDAC) in human spermatozoa.

Voltage-dependent anion channels (VDAC), also known as mitochondrial porins, are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures. VDAC allow the passage of the metabolites across the mitochondrial outer membrane, and are involved in metabolite transport and signal transduction. Several recent studies have indicated the important roles of VDAC in maintaining normal structure and motility of mammalian spermatozoa. To study the expression and localization of VDAC in human spermatozoa, different experimental approaches were applied: (1) specific primers were designed and VDAC gene sequences were cloned by PCR amplification from human testis cDNA library; (2) recombinant VDAC proteins were produced in the expression vector Escherichia coli BL21 (DE3); (3) human sperm VDAC proteins were extracted, separated and analyzed by Western blotting; (4) the localization of VDAC in human spermatozoa were detected using immunofluorescence. The three gene sequences and recombinant VDAC proteins were obtained, respectively. VDAC proteins were detected to be located in human spermatozoa, especially in sperm flagella. Our study elucidated for the first time that VDAC were synthesized and secreted at the testis level and eventually became an integral part of sperm proteins.

Role of the kinesin neck region in processive microtubule-based motility.

Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin-GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.