Emergence of asymmetry from an initially symmetrical state is a universal transition in Nature. Living organisms show striking asymmetries at the molecular, cellular, tissular and organismal level. However, whether and how multilevel asymmetries are related remains unclear. Here, we show that Drosophila Myosin 1D (Myo1D) and Myosin 1C (Myo1C) are sufficient to generate de novo directional twisting of cells, single organs or the whole body in opposite directions. We show that directionality lies in the Myosins’ motor domain and is swappable, and that Myo1D powers gliding of actin filaments in circular, counterclockwise paths in vitro. Altogether, our results reveal the molecular motor Myo1D as a chiral determinant, sufficient to break symmetry at all biological scales through chiral interaction with the actin cytoskeleton.

We report here the discovery and characterization of Ehp, a new secreted Staphylococcus aureus protein that potently inhibits the alternative complement activation pathway. Ehp was identified through a genomic scan as an uncharacterized secreted protein from S. aureus, and immunoblotting of conditioned S. aureus culture medium revealed that the Ehp protein was secreted at the highest levels during log-phase bacterial growth. The mature Ehp polypeptide is composed of 80 residues and is 44% identical to the complement inhibitory domain of S. . Further molecular level details of the Ehp/C3d interaction were revealed by solving the 2.7-Å crystal structure of an Ehp⅐C3d complex in which the low affinity site had been mutationally inactivated. Ehp potently inhibited C3b deposition onto sensitized surfaces by the alternative complement activation pathway. This inhibition was directly related to Ehp/C3d binding and was more potent than that seen for Efb-C. An altered conformation in Ehp-bound C3 was detected by monoclonal antibody C3-9, which is specific for a neoantigen exposed in activated forms of C3. Our results suggest that increased inhibitory potency of Ehp relative to Efb-C is derived from the second C3-binding site in this new protein.

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease and has been associated with a variety of neuropathological processes. ApoE4 C-terminally truncated forms have been found in brains of Alzheimer's disease patients. Structural rearrangements in apoE4 are known to be key to its physiological functions. To understand the effect of C-terminal truncations on apoE4 lipid-free structure, we produced a series of recombinant apoE4 forms with progressive C-terminal deletions between residues 166 and 299. Circular dichroism measurements show a dramatic loss in helicity upon removal of the last 40 C-terminal residues, whereas further truncations of residues 203-259 lead to recovery of helical content. Further deletion of residues 186-202 leads to a small increase in helical content. Thermal denaturation indicated that removal of residues 260-299 leads to an increase in melting temperature but truncations down to residue 186 did not further affect the melting temperature. The progressive C-terminal truncations, however, gradually increased the cooperativity of thermal unfolding. Chemical denaturation of the apoE4 forms revealed a two-step process with a clear intermediate stage that is progressively lost as the C-terminus is truncated down to residue 230. Hydrophobic fluorescent probe binding suggested that regions 260-299 and 186-202 contain hydrophobic sites, the former being solvent accessible in the wild-type molecule and the latter being accessible only upon truncation. Taken together, our results show an important but complex role of apoE4 C-terminal segments in secondary structure stability and unfolding and suggest that interactions mediated by the C-terminal segments are important for the structural integrity and conformational changes of apoE4. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 June 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptApolipoprotein E (apoE) 1 is an important protein of the lipid transport system that has an indisputable role in atherosclerosis, dyslipidemia, and Alzheimer's disease (AD) (1,2). ApoE, expressed in liver, brain, and other tissues, has three common isoforms (apoE2, apoE3, apoE4) in the general population, each differing in the amino acid positions 112 and 158 (3,4). ApoE3, the most common form, contains cysteine and arginine, respectively, whereas apoE2 has two cysteine residues and apoE4 has two arginine residues at these positions. ApoE4 has been associated with a variety of neuropathological processes, including AD (2). ApoE4 is a major genetic risk factor for AD since 40% of all patients have at least one ε4 allele (5). Being homozygous for the ε4 allele increases the risk of AD 4-fold and lowers the age of onset of late-onset AD (5). Recent studies have shown that apoE4 is also associated with carotid atherosclerosis and is a significant risk factor for coronary heart disease (6,7).ApoE contains 299 residues and in the lipid-free state is folded into two independent structu...

Summary Class-I myosins are molecular motors that link cellular membranes to the actin cytoskeleton and play roles in membrane tension generation, membrane dynamics, and mechano-signal transduction [1]. The widely expressed myosin-Ic (myo1c) isoform binds tightly to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) via a pleckstrin homology domain located in the myo1c tail, which is important for its proper cellular localization [2–4]. In this study, we found that myo1c can power actin motility on fluid membranes composed of physiological concentrations of PtdIns(4,5)P2, and that this motility is inhibited by high concentrations of anionic phospholipids. Strikingly, this motility occurs along curved paths in a counterclockwise direction (i.e., the actin filaments turn in leftward circles). A biotinylated myo1c construct containing only the motor domain and the lever arm anchored via streptavidin on a membrane containing biotinylated lipid can also generate asymmetric motility, suggesting the tail domain is not required for the counterclockwise turning. We found that the ability to produce counterclockwise motility is not a universal characteristic of myosin-I motors, as membrane-bound myosin-Ia (myo1a) and myosin-Ib (myo1b) are able to power actin gliding, but the actin gliding has no substantial turning bias. This work reveals a possible mechanism for establishing asymmetry in relationship to the plasma membrane.

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional singlebead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which has implications for a range of cellular activities, including cell division and organelle transport. SIGNIFICANCE Kinesins are cytoskeletal motors that transport cargos along microtubules. Single-bead, optical-trapping assays have been used to show that the speed and run length of kinesin are affected by mechanical load. We found that this widely used assay introduces a vertical force on the motor not accounted for in previous experiments, and this force decreases kinesin's attachment duration. Using an assay geometry that minimizes vertical forces, we found that a 180 change in the azimuthal position between a pair of net opposing forces applied between kinesin and microtubules can lead up to a 10-fold increase in the kinesin attachment duration. These results reveal a previously unknown versatility of kinesin's load-bearing capacity, which has implications for the physiological roles of kinesin.

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Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos 1 . Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors and microtubule associated proteins 2-4 . Here we report on a novel regulatory mechanism of kinesin's load-bearing capacity by forces across the microtubule track. Using optical tweezers and the three-bead assay 5,6 to specifically apply forces parallel to the long-axis of the microtubule, we found that the median attachment duration between kinesin and microtubules under opposing forces is up to 10-fold longer than observed previously using the more conventional single-bead assay, which is likely due to vertical forces imposed by the single bead 7 . Using the threebead assay, we also found that not all the protofilaments are equivalent interacting substrates for kinesin and that the median attachment duration of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (< 0.2 s) to a persistent motor that sustains attachment (> 3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by offaxis forces and forces across the microtubule lattice which has implications for a range of cellular activites including cell division and organelle transport.

Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoinositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c) is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment lifetime of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98% phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250 and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3-2.5 pN), with rates exceeding 360 s(-1) at 2.5 pN. Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to membranes during multiple ATPase cycles.