Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail.     

Vinculin controls focal adhesion formation by direct interactions with talin and actin

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.     

Restrictase BamHI, HindII, EcoRI and SmaI mapping of hybrid lambda-phi 80 phages with recombination in the structural gene region

Hybrids lambda H lambda T80 with recombination in the region of structural genes have lambda head and phi 80 tail genes. In this paper the molecular structure of 5 independently isolated hybrids was established using restriction endonucleases. It has been shown that all of them have a recombinant head or tail. A deletion of 4,8% lambda was demonstrated in the immunity region of phi 80vir phage. Co-ordinates of restriction sites for BamHI, HindIII, EcoRI and SmaI restriction endonucleases on phi 80 DNA were calculated.     

Morphology and Physical Properties of Staphylococcus Bacteriophage P11-M15

The Group B Staphylococcus phage P11-M15 is shown to be 51% protein and 49% deoxyribonucleic acid (DNA). The intact virion has a molecular weight of 66.7 x 10(6) daltons. The purified viral DNA has a molecular weight of 32.7 x 10(6) daltons. The intact virion is shown to be composed of a polyhedral head which is attached at one of its vertices to a flexible tail having helical symmetry. The tail structure is terminated by a complex baseplate which has sixfold symmetry. The virion contains a single molecule of double-stranded DNA which has no apparent single-strand nicks or single-stranded terminal redundancies.     

Molecular structure of elongated forms of electric eel acetylcholinesterase.

Molecular forms of acetylcholinesterase extracted from fresh electric organ tissue of the electric eel are elongated structures in which a multi-subunit head is connected to a fibrous tail. The principal form, 18 S acetylcholinesterase, is of molecular weight approximately 1,050,000, contains about 12 catalytic subunits in its head, has a tail approximately 500 Å long, and aggregates reversibly at low ionic strength. Trypsin converts it to an 11 S globular tetramer devoid of the tail and lacking the capacity to aggregate in low-salt solutions.Amino acid analysis shows that elongated forms of acetylcholinesterase contain significant amounts of hydroxyproline and hydroxylysine, characteristic components of collagen, which are absent from 11 S acetylcholinesterase.Collagenase converts 18 S acetylcholinesterase to a 20 S form which no longer aggregates in low salt. Purified 20 S acetylcholinesterase has about half the hydroxyproline and hydroxylysine contents of the 18 S enzyme, and physicochemical measurements indicate the formation of a more symmetrical molecular structure without marked reduction in molecular weight.Sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reducing agent shows that in 18 S acetylcholinesterase half the catalytic subunits are present as dimers linked by disulfide bonds. The remaining subunits migrate as larger molecular species which contain significant amounts of hydroxylysine, are specifically modified by collagenase and are converted to dimers and monomers by trypsin.Sodium dodecyl sulfate/acrylamide gel electrophoresis with reducing agent reveals, in 18 S acetylcholinesterase, two polypeptides of molecular weights 45,000 and 47,000 which are absent in the 11 S tetramer. They are readily digested by collagenase under conditions which do not affect the catalytic subunits, with concomitant formation of a new 30,000 polypeptide.The above data can be rationalized by a model in which 18 S acetylcholinestorase contains three subunit tetramers, each linked by disulfides to one strand of a collagen triple helix. Sodium dodecyl sulfate detaches those subunit dimers which are not covalently linked to the tail; trypsin attacks the distal portion of the collagen triple helix releasing discrete tetramers, and collagenase specifically attacks the triple helix near its midpoint, producing a shortened structure in which the residual tail still holds the tetramers together, but destroying the capacity for self-association at low ionic strength. This latter property may be related to the postulated role of the tail in anchoring acetylcholinesterase to the fibrillar matrix of the basement membrane.     

Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection

The majority of known bacteriophages have long noncontractile tails (Siphoviridae) that serve as a pipeline for genome delivery into the host cytoplasm. The tail extremity distal from the phage head is an adsorption device that recognises the bacterial receptor at the host cell surface. This interaction generates a signal transmitted to the head that leads to DNA release. We have determined structures of the bacteriophage SPP1 tail before and after DNA ejection. The results reveal extensive structural rearrangements in the internal wall of the tail tube. We propose that the adsorption device-receptor interaction triggers a conformational switch that is propagated as a domino-like cascade along the 1600 A-long helical tail structure to reach the head-to-tail connector. This leads to opening of the connector culminating in DNA exit from the head into the host cell through the tail tube.     

Crystal structure of reovirus attachment protein sigma1 reveals evolutionary relationship to adenovirus fiber.

Reovirus attaches to cellular receptors with the sigma1 protein, a fiber-like molecule protruding from the 12 vertices of the icosahedral virion. The crystal structure of a receptor-binding fragment of sigma1 reveals an elongated trimer with two domains: a compact head with a new beta-barrel fold and a fibrous tail containing a triple beta-spiral. Numerous structural and functional similarities between reovirus sigma1 and the adenovirus fiber suggest an evolutionary link in the receptor-binding strategies of these two viruses. A prominent loop in the sigma1 head contains a cluster of residues that are conserved among reovirus serotypes and are likely to form a binding site for junction adhesion molecule, an integral tight junction protein that serves as a reovirus receptor. The fibrous tail is mainly responsible for sigma1 trimer formation, and it contains a highly flexible region that allows for significant movement between the base of the tail and the head. The architecture of the trimer interface and the observed flexibility indicate that sigma1 is a metastable structure poised to undergo conformational changes upon viral attachment and cell entry.     

Coliphage P1 morphogenesis: analysis of mutants by electron microscopy.

We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage.     

Effect of concentration on the aggregation of poly(γ-benzyl-L-glutamate) in dioxane: Critical concentration and maximum length for linear head to tail aggregates

Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ-benzyl-L -glutamate) in dioxane have been carried out over a range of concentration 10^?4–10^?2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (? 10^?3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration. Maximum length of linear head to tail type aggregates for poly(γ-benzyl-L -glutamate) in dioxane as determined from these results correspond to an α-helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.     

Effect of the sugar moiety on complex formation of cycloamyloses with p-nitrophenyl glycosides

Circular diochroism (CD) spectra of four p-nitrophenyl glycosides and their cycloamylose complexes were investigated at various concentrations of cycloamylose and at temperatures ranging from 20 to 60°C. The CD spectra of p-nitrophenyl glycosides changed remarkably in the presence of cycloamyloses, and significant differences in spectral shape and intensity were observed between the cyclohexaamylose complex and the cycloheptaamylose complex. The difference CD spectra between the free guest and its complex indicates that the nitrophenyl group is included in the cycloamylose cavity but its disposition is different between the complexes with cyclohexaamylose and cycloheptaamylose. Values of enthalpy and entropy of the cyclohexaamylose complex are considerably larger than those of the corresponding cycloheptaamylose complex, although the free energy differs only slightly. It is suggested that the nitrophenyl group is more loosely bound to the cycloheptaamylose cavity than to the cyclohexaamylose cavity, and has much more flexibility in its disposition.     

Structure of native and chloroform-methanol-treated mycobacteriophage R1.

The morphologies of native and chloroform-methanol-treated mycobacteriophage R1 were compared by electron microscopy, utilizing three negative stains. R1 was determined to be a complex phage. The head appears as an elongated cylinder with a pointed end (93 +/- 3 by 42 +/- 3 nm) constructed from an orderly arrangement of capsomeres. The phage tail measures 209 +/- 11 by 11 +/- 1 nm and possesses a striated surface with two base plates at its distal end. Treatment of R1 with chloroform-methanol resulted in disruption of both the head and tail structures and was accompanied by loss of infectivity. However, because no likely lipid-containing structure was observed in native phages, there is the possibility that the mechanism of chloroform-methanol inactivation is something other than lipid extraction.     

Speract induces calcium oscillations in the sperm tail

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.     

Visualisation by low-angle shadowing of the leucocyte-common antigen. A major cell surface glycoprotein of lymphocytes.

The leucocyte-common antigen (L-CA) from rat thymocytes is a cell surface glycoprotein of 180 000 apparent mol. wt. with an 80-kd cytoplasmic domain. This paper reports the molecular dimensions of the molecule visualised by electron microscopy after low-angle shadowing. The L-CA monomer consists of a globular head region of approximately 12 nm diameter and a short tail approximately 18 nm long. In deoxycholate both monomers and multimers are seen with aggregation occurring at the head groups. When the detergent is removed, larger clusters are formed with tails extending from a central aggregate. A 100-kd tryptic fragment of L-CA that is known to include the extracellular parts of the molecule also exists in monomer and multimer forms and is seen to have a rod-like structure of length 28 nm without evidence of the head group. Altogether the data indicate that the rod-like structure is found outside the cell and that the extra sequence that forms the head is inside. The tryptic fragment is likely to be derived by cleavage after the transmembrane sequence.     

Molecular characterization of the Salmonella enterica serovar Typhi Vi-typing bacteriophage E1

Some bacteriophages target potentially pathogenic bacteria by exploiting surface-associated virulence factors as receptors. For example, phage have been identified that exhibit specificity for Vi capsule producing Salmonella enterica serovar Typhi. Here we have characterized the Vi-associated E1-typing bacteriophage using a number of molecular approaches. The absolute requirement for Vi capsule expression for infectivity was demonstrated using different Vi-negative S. enterica derivatives. The phage particles were shown to have an icosahedral head and a long noncontractile tail structure. The genome is 45,362 bp in length with defined capsid and tail regions that exhibit significant homology to the S. enterica transducing phage ES18. Mass spectrometry was used to confirm the presence of a number of hypothetical proteins in the Vi phage E1 particle and demonstrate that a number of phage proteins are modified posttranslationally. The genome of the Vi phage E1 is significantly related to other bacteriophages belonging to the same serovar Typhi phage-typing set, and we demonstrate a role for phage DNA modification in determining host specificity.     

The small heat shock protein ODF1/HSPB10 is essential for tight linkage of sperm head to tail and male fertility in mice

Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.     

Ultrastructure of the spermatozoon of Megaselia scalaris Loew (Diptera:Brachycera:Cyclorrhapha:Phoridea:Phoridae)

When viewed by scanning electron microscopy (SEM), the spermatozoon of the phorid dipteran Megaselia scalaris appears threadlike, lacking distinct head and tail areas. These areas can be observed, however, in appropriately stained material. Measurements of Feulgen-stained material reveal average lengths of the head, tail, and total cell of 18.7, 128.7, and 147.4 μm, respectively. When tested for sulfhydryl and disulfide groups, the head displays only disulfide groups. Transmission electron microscopy (TEM) reveals 12 different regions: three (1–3) in the head, four (9–12) in the tail, and five (4–8) in a short zone of overlap between the head and tail. Most of the cell lies in regions 9 and 11 of the tail and 3 of the head, accounting for, respectively, 37.3%, 45.7%, and 11.2% of the total length. A tubelike acrosome indents the anterior end of the nucleus. The tail originates asymmetrically in relation to the long axis of the cell as a peglike structure associated with the dorsolateral region of the nucleus. No centriole is visible, and the nucleus has a notched appearance in longitudinal sections. Two mitochondrial derivatives and an axoneme displaying a 9+9+2 microtubule configuration and ATPase activity extend throughout most of the tail. In regions 9 and 10, an asymmetrically arranged accessory body is also present. Features having possible taxonomic utility include the asymmetrically arranged accessory body, the size and shape of the acrosome, and the notched appearance of the nucleus. The present report is apparently the first to describe the spermatozoon of a cyclorrhaphous dipteran which is not a member of the Schizophora.     

Structure of Bacillus subtilis Bacteriophage φ25 and φ25 Deoxyribonucleic Acid

The structure of Bacillus subtilis bacteriophage phi25 and phi25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of phi25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of phi25 is 130 nm in length and includes a large, complex tail plate. Phage phi25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of phi25 DNA contain numerous random interruptions. Chemical analysis of phi25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.     

Crystal structure of Mdm12 reveals the architecture and dynamic organization of the ERMES complex

The endoplasmic reticulum–mitochondria encounter structure (ERMES) is a protein complex that plays a tethering role in physically connecting ER and mitochondria membranes. The ERMES complex is composed of Mdm12, Mmm1, and Mdm34, which have a SMP domain in common, and Mdm10. Here, we report the crystal structure of S. cerevisiae Mdm12. The Mdm12 forms a dimeric SMP structure through domain swapping of the β1‐strand comprising residues 1–7. Biochemical experiments reveal a phospholipid‐binding site located along a hydrophobic channel of the Mdm12 structure and that Mdm12 might have a binding preference for glycerophospholipids harboring a positively charged head group. Strikingly, both full‐length Mdm12 and Mdm12 truncated to exclude the disordered region (residues 74–114) display the same organization in the asymmetric unit, although they crystallize as a tetramer and hexamer, respectively. Taken together, these studies provide a novel understanding of the overall organization of SMP domains in the ERMES complex, indicating that Mdm12 interacts with Mdm34 through head‐to‐head contact, and with Mmm1 through tail‐to‐tail contact of SMP domains.     

Structural Characterization of the Bacteriophage T7 Tail Machinery

Most bacterial viruses need a specialized machinery, called “tail,” to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well characterized member of the Podoviridae family infecting Escherichia coli, and it has a short noncontractile tail that assembles sequentially on the viral head after DNA packaging. The T7 tail is a complex of around 2.7 MDa composed of at least four proteins as follows: the connector (gene product 8, gp8), the tail tubular proteins gp11 and gp12, and the fibers (gp17). Using cryo-electron microscopy and single particle image reconstruction techniques, we have determined the precise topology of the tail proteins by comparing the structure of the T7 tail extracted from viruses and a complex formed by recombinant gp8, gp11, and gp12 proteins. Furthermore, the order of assembly of the structural components within the complex was deduced from interaction assays with cloned and purified tail proteins. The existence of common folds among similar tail proteins allowed us to obtain pseudo-atomic threaded models of gp8 (connector) and gp11 (gatekeeper) proteins, which were docked into the corresponding cryo-EM volumes of the tail complex. This pseudo-atomic model of the connector-gatekeeper interaction revealed the existence of a common molecular architecture among viruses belonging to the three tailed bacteriophage families, strongly suggesting that a common molecular mechanism has been favored during evolution to coordinate the transition between DNA packaging and tail assembly.