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Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes., This research was supported by funds from SAF2013-49179-C2-1-R JPND_CD_FP-688- 059 (AC14/00037 ISCIII) to MCV, SIMR to KS, SAF2013-49179-C2-2-R JPND_CD_FP-688-059 (AC14/00037 ISCIII) to DVL, BFU2012-36825, S2011/BMD-2457 (Comunidad de Madrid)

Actin filaments and myosin-II are evolutionarily conserved force generating components of the contractile ring during cytokinesis. Here we show that in budding yeast actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuates actomyosin ring constriction. Deletion of myosin-II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin-II motor activity. Model simulations based on experimental measurements supports the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time irrespective of the initial ring size as originally reported for C elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to having different ploidies.

SummaryThe centromere is a specialized chromosomal structure that regulates chromosome segregation. Centromeres are marked by a histone H3 variant. In budding yeast, the histone H3 variant Cse4 is present in a single centromeric nucleosome. Experimental evidence supports several different models for the structure of centromeric nucleosomes. To investigate Cse4 copy number in live yeast, we developed a method coupling fluorescence correlation spectroscopy and calibrated imaging. We find that centromeric nucleosomes have one copy of Cse4 during most of the cell cycle, whereas two copies are detected at anaphase. The proposal of an anaphase-coupled structural change is supported by Cse4-Cse4 interactions, incorporation of Cse4, and the absence of Scm3 in anaphase. Nucleosome reconstitution and ChIP suggests both Cse4 structures contain H2A/H2B. The increase in Cse4 intensity and deposition at anaphase are also observed in Candida albicans. Our experimental evidence supports a cell-cycle-coupled oscillation of centromeric nucleosome structure in yeast.

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition., Author Summary Accurate segregation of chromosomes during mitosis is essential to prevent genetic instability and aneuploidy that lead to cancer and other diseases. Centrosomes and spindle pole bodies mediate the assembly of a microtubule-based structure known as the mitotic spindle, which physically separates chromosomes during mitosis so that the two daughter cells contain a complete copy of the genetic material as well as a spindle pole. During every cell cycle, the DNA and the spindle pole must be duplicated exactly once to ensure proper formation of a bipolar mitotic spindle. In yeast cells, the nuclear envelope does not break down, so the spindle pole must be inserted into the nuclear membrane so that it can form both the microtubules involved in the mitotic spindle and those involved in positioning of the nucleus. How a large protein complex such as the spindle pole body is inserted into the lipid layers of the nuclear membrane is not well understood. We show that the evolutionarily conserved SUN protein Mps3 is involved in spindle pole insertion into the nuclear membrane. This likely reflects a function for SUN proteins in controlling nuclear envelope structure by modulating the types of lipids that are present in the nuclear membrane.

Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and 35S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies., Author Summary Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. Two human diseases, Cornelia de Lange syndrome and Roberts syndrome, are caused by mutations in cohesin. These “cohesinopathies” are thought to be caused by gene misregulation, although the role of cohesin in transcription has been enigmatic. Here we show that mutations in cohesin are associated with reduced production of the structural RNAs that are components of the ribosome in the budding yeast Saccharomyces cerevisiae. This causes defects in protein translation, which can explain a large fraction of the gene misregulation observed. We further show similar physiology in a human Roberts syndrome cell line. We postulate that reduced translational capacity contributes to the cohesinopathies.

Protein interactions are inherently dynamic. In no system is this more true and important than in signaling pathways, where spatial and temporal control of specific protein interactions is key to signaling specificity and timing. While genetic and biochemical interactions form a necessary and important starting point for deciphering interactions among signaling components, they struggle to provide precise information of where and when interactions occur in a live cell setting. In contrast, live cell fluorescence studies such as those outlined below are able to provide quantitative information on the strength, nature, timing, and location of homotypic and heterotypic protein interactions.

Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

To measure protein interactions, diffusion properties, and local concentrations in single cells, Fluorescence Correlation Spectroscopy (FCS) is a well-established and widely accepted method. However, measurements can take a long time and are laborious. Therefore investigations are typically limited to tens or a few hundred cells. We developed an automated system to overcome these limitations and make FCS available for High Content Screening (HCS). We acquired data in an auto-correlation screen of more than 4000 of the 6000 proteins of the yeast Saccharomyces cerevisiae, tagged with eGFP and expanded the HCS to use cross-correlation between eGFP and mCherry tagged proteins to screen for molecular interactions. We performed all high-content FCS screens (HCS-FCS) in a 96 well plate format. The system is based on an extended Carl Zeiss fluorescence correlation spectrometer ConfoCor 3 attached to a confocal microscope LSM 510. We developed image-processing software to control these hardware components. The confocal microscope obtained overview images and we developed an algorithm to search for and detect single cells. At each cell, we positioned a laser beam at a well-defined point and recorded the fluctuation signal. We used automatic scoring of the signal for quality control. All data was stored and organized in a database based on the open source Open Microscopy Environment (OME) platform. To analyze the data we used the image processing language IDL and the open source statistical software package R.

Bacterial Artificial Chromosomes (BACs) are vital tools in mouse genomic analyses because of their ability to propagate large inserts. The size of these constructs, however, prevents the use of conventional molecular biology techniques for modification and manipulation. Techniques such as recombineering and Cre/Lox methodologies have thus become heavily relied upon for such purposes. In this work, we investigate the applicability of Lox variant sites for serial and/or simultaneous manipulations of BACs. We show that Lox spacer mutants are very specific, and inverted repeat variants reduce Lox reaction rates through reducing the affinity of Cre for the site, while retaining some functionality. Employing these methods, we produced serial modifications encompassing four independent changes which generated a mouse HoxB BAC with fluorescent reporter proteins inserted into four adjacent Hox genes. We also generated specific, simultaneous deletions using combinations of spacer variants and inverted repeat variants. These techniques will facilitate BAC manipulations and open a new repertoire of methods for BAC and genome manipulation.

Protein interactions are inherently dynamic. In no system is this more true and important than in signaling pathways, where spatial and temporal control of specific protein interactions is key to signaling specificity and timing. While genetic and biochemical interactions form a necessary and important starting point for deciphering interactions among signaling components, they struggle to provide precise information of where and when interactions occur in a live cell setting. In contrast, live cell fluorescence studies such as those outlined below are able to provide quantitative information on the strength, nature, timing, and location of homotypic and heterotypic protein interactions.

We present a study of reorientation dynamics of the three aromatic amino acids and their side chain models in aqueous solution. Experimentally, anisotropy decay measurements with picosecond time resolution were performed for blocked tryptophan, tyrosine, and phenyalanine and model compounds p-cresol and 3-methylindole. Computationally, rotational diffusion was modeled by molecular dynamics simulations for the three aromatic residues and their side chain models: benzene, toluene, phenol, p-cresol, indole, and 3-methylindole in explicit water. Our simulations used the CHARMM protein force field and associated TIP3P water model and tend to underestimate the rotational correlation times. However, the simulations yield several interesting qualitative insights into reorientational motions that complement the experimental measurements. The effects of substituent and temperature on reorientations of the parent compounds are well reproduced computationally. Additionally, simulations indicate strongly anisotropic reorientations for most of the studied compounds and a separation of time scales between conformational dynamics and rotational diffusion. Comparison with continuum hydrodynamic models suggests that we may consider that the blocked amino acids move under stick boundary conditions, while the dynamics for most of the model compounds falls between stick and slip conditions. Our systematic treatment of blocked amino acids, starting from the parent compounds (benzene, phenol, and indole) provides a baseline for understanding the anisotropy decay signals of more complicated peptide systems.

Maintenance of robust cell polarization is a pre-requisite for oriented growth or motility biological systems. Cdc42 is a Rho-GTPase that, once polarized, organizes the deposition of growth materials to the polarized site. Previous studies in yeast have found that after initial polarity establishment, the individual molecules of Cdc42 are rapidly exchanged between the membrane and the cytosol. Despite these rapid dynamics, the site of Cdc42 accumulation is somehow held rigid to allow for polarized growth. We show that after initial symmetry breaking, Cdc42 is dynamically maintained at the polar cap through two recycling mechanisms: actin-mediated transport / endocytosis and cytosolic recycling through the Rho-GDP dissociation inhibitor (GDI) Rdi1. However, the mechanism by which the spatial relationship of these dual recycling pathways is controlled is unclear. We combined detailed live cell fluorescence measurements, including FRAP, iFRAP and fluorescence cross-correlation spectroscopy, with a thorough mathematical model to examine how dual recycling pathways of Cdc42 in yeast work together to shape the Cdc42 membrane distribution. We find that in order to recapitulate the steady-state membrane distribution of WT Cdc42, the dual recycling pathways must employ overlapping delivery windows of similar size. Interestingly, the modeling of live cell fluorescence data reveals that Rdi1-mediated tuning of a single dynamic parameter, internalization rate inside the delivery window, is sufficient to explain differences in the Cdc42 cap distribution between yeast cells destined for different morphogenic fates. These changes in Cdc42 cap distribution are correlated to the observed morphogenic differences under these conditions.

We have developed an automated fluorescence cross-correlation screen to detect molecular interactions in yeast. Using fluorescence cross-correlation spectroscopy (FCCS) positive control strains containing linked eGFP and mCherry proteins could be reproducibly distinguished from negative controls with independently diffusing populations of the two fluorophores. Transmitted light images were acquired in parallel with FCCS measurements to determine protein localization and cell health. Data was taken in a 96-well format on a commercially available microscope with the addition of a ConfoCor3 (Carl Zeiss Jena GmbH, Germany). Custom software controlled navigation of the 96-well plates, detection of yeast cells, and selection of cellular regions for taking FCCS measurements, making the screen adaptable for larger scale experiments. Using this method, proteins of particular interest can be rapidly screened against large portions of the proteome to uncover their contribution to the function of a complex network of proteins.

Two-photon scanning fluorescence microscopy has become a powerful tool for imaging living cells and tissues. Most applications of two-photon microscopy employ a Ti:sapphire laser excitation source, which is not readily portable or rapidly tunable. This work explores the use of two-photon fiber laser excitation (TP-FLEX) as an excitation source for scanning two-photon microscopy. We have further demonstrated the use of a photonic crystal fiber (PCF) for facile tuning of the excitation wavelength over the range from 810 nm to 1100 nm. We generated two-photon fluorescence images at excitation wavelengths from 850 nm to 1100 nm detected on a scanning-stage microscope. By PCF wavelength tuning the dye BODIPY fl was selectively excited at 1000 nm whereas MitoTracker red was excited preferentially at 1100 nm. We discuss the potential for fiber laser sources coupled with PCF wavelength tuning as an attractive tunable excitation source for two-photon scanning fluorescence microscopy.

We demonstrate the calculation of particle brightness and concentration from fluorescence-fluctuation photon-counting statistics using an electron-multiplied charge-coupled device (EMCCD) camera. This technique provides a concentration-independent measure of particle brightness in dynamic systems. The high sensitivity and highly parallel detection of EMCCD cameras allow for imaging of dynamic particle brightness, providing the capability to follow aggregation reactions in real time. A critical factor of the EMCCD camera is the presence of nonlinearity at high intensities. These nonlinearities arise due to limited capacity of the CCD well and to the analog-to-digital converter maximum range. However, we show that the specific camera we used (with a 16-bit analog-to-digital converter) has sufficient dynamic range for most microscopy applications. In addition, we explore the importance of camera timing behavior as it is affected by the vertical frame transfer speed of the camera. Although the camera has microsecond exposure time for illumination of a few pixels, the exposure time increased to milliseconds for full-field illumination. Finally, we demonstrate the ability of the technique to follow concentration changes and measure single-molecule brightness in real time in living cells. © 2008 by the Biophysical Society.

The results described here demonstrate that single-molecule fluorescence spectroscopy of molecules freely diffusing in solution can yield unique information about both the dynamics and conformations of proteins. We used single-molecule FRET to detect the dynamics in CaM on the timescales of 100’s of microseconds and a few milliseconds by cross-correlation analysis of the donor and acceptor signals. In addition, analysis of the distribution of donor-acceptor distances by single-molecule FRET demonstrates the presence of three distinct conformational substates of CaM in solution. Together these results paint a picture of a protein that is dynamic and flexible, accessing a range of distinct conformation substates in solution.

Differential effects of ligand binding on local and global fibroblast growth factor-10 (FGF-10) flexibility and stability have been investigated utilizing a variety of experimental and computational techniques. Normal mode analysis was used to predict the low frequency motions and regional flexibility of FGF-10. Similarly, regional variations in local folding/unfolding equilibria were characterized with the COREX/BEST algorithm. Experimental adiabatic and isothermal compressibilities of FGF-10 alone and in the presence of polyanions are compared. Furthermore, the effect of polyanions on the coefficient of thermal expansion is compared. Measurements of density, heat capacity, compressibility, and expansibility were combined to calculate experimentally determined volume and enthalpy fluctuations. Global effects of polyanions on FGF-10 flexibility, thermodynamic fluctuations, and hydration vary depending on the size and charge density of the polyanion. Local effects of polyanions were investigated utilizing time-resolved fluorescence spectroscopy and red edge excitation spectroscopy (REES). Increased rigidity of the protein matrix or an increased solvent response surrounding the Trp residues is observed in the presence of polyanions. Similarly, time-resolved spectroscopy reveals increased ground state heterogeneity and increased dipole relaxation on the time scale of fluorescence for FGF-10 in the presence of polyanions. These polyanions increase heterogeneity, global flexibility, and fluctuations while increasing the melting temperature (Tm) of FGF-10.

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.

A rapid, homogeneous aptamer-based bioanalysis is reported for the sensitive detection of immunoglobulin E (IgE) using fluorescence polarization (FP). 5'-End-labeled D17.4 DNA aptamer was used for IgE detection based on the anisotropy differences of the labeled ligand. Two different fluorophores, fluorescein and Texas Red, were used to analyze IgE in the low-nanomolar range with high specificity. Measurable anisotropy changes were observed with a short equilibration time. Analysis of the binding data reveals a possible cooperative binding process in solution. The nature of the fluorophore clearly influences the sensitivity of the analysis more than the tether length used for the dye conjugation. The local fluorophore motion is seen to influence the sensitivity of the FP probe significantly. Texas Red is seen to be relatively more sensitive for this approach and has apparently favorable dye-DNA interactions, and a limit of detection of 350 pM was obtained. Significant temperature dependence of the FP responses has been observed in this work. Ionic composition of the binding buffer also influences the assay sensitivity. The results confirm the promise and potential of similar homogeneous assays for aptamer-based bioanalysis.

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.

We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or “aptamer” designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5′-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.

The ability of peptides to form biologically active conformations that bind to receptors is governed by their dynamics and their propensity to form stable structures. Such factors are consequently important in the design of peptide drugs. Moreover, the stability of such peptides depends on interactions of the peptide with the surrounding matrix. In this article, we study the effect of the polymer poly(vinyl pyrrolidone) (PVP) on the mobility and orientational dynamics of tyrosine and a model peptide, Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA) in glycerol-water solutions. Orientational dynamics are studied experimentally by time-resolved fluorescence anisotropy decays of tyrosine. The presence of PVP leads to the possibility of a distribution of environments for the peptide. The orientational dynamics of tyrosine show that the probe molecule experiences two very different environments. In one, tyrosine rotational motion is weakly coupled to PVP, while in the other, tyrosine interacts strongly with PVP leading to much slower rotational times. The dynamics of VYPNGA are more complex. Fast intramolecular, localized reorientations of the tyrosine are detected. The temperature dependence of the reorientational dynamics of the tyrosine side chain reveal that these motions are shielded from solvent friction. In contrast, global motions of the peptide are severely restricted by PVP, suggesting the ability of the polymer to restrict peptide mobility.

We use in vivo fluorescence correlation spectroscopy (FCS) and high sensitivity quantitative confocal imaging to demonstrate a dramatic structural rearrangement of the yeast centromere in early anaphase. using FCS, we show that the yeast centromeric histone, Cse4, is present in the early kinetochore at a stoichiometry of one protein per chromosome and doubles to two copies during the structural change. This transition reconciles the controversy between previous results observing both forms in vitro. We have confirmed this result by quantifying nuclear pore complex stoichiometries which are well known. Fluorescence recovery after photobleaching (FRAP) experiments further confirm our result, demonstrating centromeric protein addition specifically during anaphase. Fluorescence resonance energy transfer (FRET) experiments also confirm the result, demonstrating homotypic interactions only in late anaphase. The yeast centromere is an important model complex whose functions are central to many important biological processes. These results have revolutionized our understanding of these basic processes and provide fertile ground for future studies of the kinetochore. Our method demonstrates a unique modality for FCS and quantitative imaging and will likely provide much needed insight into biologically important structures in the future.

In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles., Author Summary Proper chromosome segregation during cell division is essential. Missegregation of mitotic chromosomes leads to cell death or cancer, and chromosome missegregation during meiosis leads to miscarriage and birth defects. Cells utilize a bipolar microtubule-based structure known as the meiotic or mitotic spindle to segregate chromosomes. Because proper bipolar spindle formation is critically important for chromosome segregation, cells have many redundant mechanisms to ensure that this structure is properly formed. In most animal cells, centrosomes containing γ-tubulin protein complexes help organize and shape the bipolar spindle. Since meiosis I spindles in oocytes lack centrosomes, the mechanisms by which a meiotic bipolar spindle is assembled are not fully understood. In Drosophila oocytes it was not clear whether γ-tubulin played a role in bipolar spindle assembly or if it was even present on the meiotic spindle. We demonstrate that γ-tubulin plays vital roles in bipolar spindle formation and maintenance, as well as in aligning the chromosomes on the oocyte spindle. Additionally, we show that γ-tubulin is present on the bipolar spindle in Drosophila oocytes. More importantly, we demonstrate that γ-tubulin plays a critical role in the formation of the kinetochore microtubules that are required to properly orient chromosomes on the meiotic spindle.

Multiphoton microscopy (MPM) holds promise as a noninvasive imaging technique for characterizing collagen structure, and thus mechanical properties, through imaging second harmonic generation (SHG) and two-photon fluorescence in engineered and real connective tissues. Controlling polymerization pH to manipulate collagen gel microstructure, we quantified pore and fiber dimensions using both standard methods and image correlation spectroscopy (ICS) on MPM, scanning electron, and darkfield microscopy images. The latter two techniques are used to confirm microstructural measurements made from MPM images. As polymerization pH increased from 5.5 to 8.5, mean fiber diameter decreased from 3.7 +/- 0.7 microm to 1.6 +/- 0.3 microm, the average pore size decreased from 81.7 +/- 3.7 microm(2) to 7.8 +/- 0.4 microm(2), and the pore area fraction decreased from 56.8% +/- 0.8% to 18.0% +/- 1.3% (measured from SHG images), whereas the storage modulus G' and loss modulus G'', components of the shear modulus, increased approximately 33-fold and approximately 16-fold, respectively. A characteristic length scale measured using ICS, W(ICS), correlates well with the mean fiber diameter from SHG images (R(2) = 0.95). Semiflexible network theory predicts a scaling relationship of the collagen gel storage modulus (G') depending upon mesh size and fiber diameter, which are estimated from SHG images using ICS. We conclude that MPM and ICS are an effective combination to assess bulk mechanical properties of collagen hydrogels in a noninvasive, objective, and systematic fashion and may be useful for specific in vivo applications.

SummaryA long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, we postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. Here, we found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24 hr. However the mutant flies failed to stabilize memory beyond 48 hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory.

SummaryAsymmetric inheritance of protein aggregates in budding yeast is a fascinating yet controversial area of aging research. A recent study demonstrates that unfolded protein aggregates are confined to the mother by tethering to organelles rather than retrograde transport.

In living cells, protein complexes are dynamic structures that control many distinct aspects of cell behavior. Addressing the complexity of protein function requires a comprehensive understanding of subcellular localization and protein stoichiometry. Fluorescence microscopy provides a high contrast method to determine specific localizations of individual proteins contained within living cells. Determining localizations of protein pairs is made difficult by inherent photobleaching, cross-talk, and autofluorescence contained within the sample being imaged. Traditionally, to separate fluorescent proteins (FPs) with overlapping spectra, techniques such as linear unmixing and fluorescence lifetime imaging (FLIM) have been used. Such techniques rely on complex microscope configurations and often require a compromise in signal to noise ratios. In these studies we use phase information derived from optical lock in detection (OLID) (Mao et al. Biophys J. 2008 Jun;94(11):4515-2) to separate the photoswitchable FPs Dronpa-1, Dronpa-2, and Dronpa-3 from GFP and autofluorescence with no effect in signal to noise. Additionally, relative stochiometric ratios between each of the Dronpa variants contained within microinjected zebrafish embryos were obtained with a standard confocal microscope.

High-content screening fluorescence correlation spectroscopy (HCS-FCS) determines diffusion properties, local concentrations and molecular interactions of biologically relevant proteins in thousands of individual S. cerevisiae cells. We automated one color auto-correlation fluorescence spectroscopy to investigate the properties of the yeast proteome. We also used two-color cross-correlation for interaction screens.For the proteome wide one-color yeast screen, we investigated more than 4000 of the approximately 6000 proteins known in S. cerevisiae. This resulted in over 200,500 measurements on more than 50,000 individual cells. This amount of data required automated tools for data quality control and analysis. Our pipeline started with the evaluation of transmitted light images of the measured cells. We extracted features from these images and trained a support vector machine (SVM) to classify them into healthy yeast cells and samples we wanted to exclude from further analysis. A similar approach enabled us to classify raw fluctuation data taken from the remaining cells and the auto-correlation curves we derived from them. As training sets, we used measured and simulated data.To analyze the correlation curves obtained from the fluctuation data, we fitted different diffusion models. We used mixed-effects models to extract averaged fit parameters for the same protein measured in multiple cells. To select the most appropriate diffusion model we used Akaike's ‘An Information Criterion’ (AIC).This approach not only allows the analysis of large data-sets as they occur in our HCS-FCS experiments and camera based FCS described in literature, but has the additional advantage of reducing the human bias.

We demonstrate a new method for measurements of mobility dependent protein oligomerization using higher order fluorescence correlation cumulants. Fluorescence intensity distribution methods including fluorescence cumulant and moment analysis have been successfully used in recent years to analyze oligomerization phenomena. The extension of such methods to treat analysis at different binning times allows for the analysis of mobility dependent oligomerization. Nevertheless, the analysis of time binned distributions is mathematically complex and depends strongly on detector characteristics such as afterpulsing and dead time. Here we develop an equivalent method treating traditional correlation functions as bivariate fluorescence cumulants. Doing so brings the power of the cumulant analysis to bear on the measurement of mobility dependent oligomerization while maintaining the mathematical simplicity of the correlation functions that are the standard within fluorescence correlation spectroscopy. We use this technique to show that the low mobility pool of intracellular EGFP-cdc-42 in living yeast cells is on average a dimer while the high mobility pool is monomeric. Examination of mutant yeast strains suggests that the low mobility pool is associated with recycling vesicles, providing an explanation for both the slow diffusion as well as the oligomeric state of this species. This technique could be easily extended to other proteins in the yeast genome that demonstrate heterogeneous mobility.

We use spatio-temporal correlation methods with transmitted light microscopy to demonstrate a novel pattern of internal-to-cortical circular cytoplasmic flow in meiosis II mouse oocytes. This flow is responsible for poising the spindle near the cortex for completion of meiosis II and successful oocyte fertilization. We show that this flow is driven by a balance of Arp2/3-complex-induced actin nucleation at the cortex and myosin-II-driven contraction of the cortex. STICS analysis of EGFP-Utrophin-labeled actin fibers shows cortical outflows with velocities consistent with those required to drive the cytoplasmic streaming. Inhibition of the Arp2/3-complex with CK-666 reverses this flow and leads to internal movement of the spindle away from the cortex. Interestingly, neither inhibition of actin polymerization, myosin-II contraction, nor microtubule depolymerization resulted in spindle internalization or large changes in cytoplasmic streaming. Kymograph analysis of the cortex stained with fluorescent Concavalin A demonstrates cortical contraction that is eliminated upon inhibition of myosin-II. Combined inhibition of myosin and Arp2/3-complex results in elimination of reversed cytoplasmic streaming.View Large Image | View Hi-Res Image | Download PowerPoint Slide