12.4: Microtubules - Biology

Microtubules are made up of two equally distributed, structurally similar, globular subunits: α and β tubulin. Like microfilaments, microtubules are also dependent on a nucleotide triphosphate for polymerization, but in this case, it is GTP.

Microtubule stability is temperature-dependent: if cooled to 4°C, microtubules fall apart into αβ-tubulin heterodimers. Warmed back up to 37°C, the tubulin repolymerizes if there is GTP available.

Another similarity is that microtubules have a polarity in which the (-) end is far less active than the (+) end. However, unlike the twisted-pair microfilaments, the microtubules are mostly found as large 13-stranded (each strand is called a protofilament) hollow tube structures. Also, the α and β tubulin used for building the microtubules not only alternate, but they are actually added in pairs. Both the α-tubulin and β-tubulin must bind to GTP to associate, but once bound, the GTP bound to α-tubulin does not move. On the other hand, GTP bound in the β-tubulin may be hydrolyzed to GDP. GDP-bound αβ-dimers will not be added to a microtubule, so similar to the situation with ATP and g-actin, if the tubulin has GDP bound to it, it must first exchange it for a GTP before it can be polymerized. Although the affinity of tubulin for GTP is higher than the affinity for GDP, this process is usually facilitated by a GEF, or guanine nucleotide exchange factor. As the signal transduction chapter will show in more detail, this type of nucleotide exchange is a common mechanism for activation of various biochemical pathways.

Again like actin, the tubulin itself has enzymatic activity, and over time, the GTPase activity hydrolyzes the GTP to GDP and phosphate. This changes the attachment between β-tubulin of one dimer and the α-tubulin of the dimer it is stacked on because the shape of the subunit changes. Even though it isn’t directly loosening its hold on the neighboring tubulin, the shape change causes increased stress as that part of the microtubule tries to push outward. This is the basis of a property of microtubules known as dynamic instability. If there is nothing to stabilize the microtubule, large portions of it will fall apart. However, as long as new tubulin (which will have GTP bound) is being added at a high enough rate to keep a section of low-stress “stable”-conformation microtubule (called the GTP cap) on top of the older GDP-containing part, then it stabilizes the overall microtubule. When new tubulin addition slows down, and there is only a very small or nonexistent cap, then the microtubule undergoes a catastrophe in which large portions rapidly break apart. Note that this is a very different process than breakdown by depolymerization, which is the gradual loss of only a few subunits at a time from an end of the microtubule. Depolymerization also occurs, and like with actin, is determined partially by the relative concentrations of free tubulin and microtubules.

From a physical standpoint, the microtubule is fairly strong, but not very flexible. A microfilament will flex and bend when a deforming force is applied (imagine the filament anchored at the bottom end standing straight up, and something pushing the tip to one side). The microtubule in the same situation will bend only slightly, but break apart if the deforming force is sufficient. There is, of course, a limit to the flexibility of the microfilament and eventually, it will also break. Intermediate filaments are slightly less flexible than the microfilaments, but can resist far more force that either microfilaments or microtubules.

Cell (biology)

The cell (from Latin cella, meaning "small room" [1] ) is the basic structural, functional, and biological unit of all known organisms. Cells are the smallest units of life, and hence are often referred to as the "building blocks of life". The study of cells is called cell biology, cellular biology, or cytology.

Cells consist of cytoplasm enclosed within a membrane, which contains many biomolecules such as proteins and nucleic acids. [2] Most plant and animal cells are only visible under a light microscope, with dimensions between 1 and 100 micrometres. [3] Electron microscopy gives a much higher resolution showing greatly detailed cell structure. Organisms can be classified as unicellular (consisting of a single cell such as bacteria) or multicellular (including plants and animals). [4] Most unicellular organisms are classed as microorganisms.

The number of cells in plants and animals varies from species to species it has been estimated that humans contain somewhere around 40 trillion (4×10 13 ) cells. [a] [5] The human brain accounts for around 80 billion of these cells. [6]

Cells were discovered by Robert Hooke in 1665, who named them for their resemblance to cells inhabited by Christian monks in a monastery. [7] [8] Cell theory, first developed in 1839 by Matthias Jakob Schleiden and Theodor Schwann, states that all organisms are composed of one or more cells, that cells are the fundamental unit of structure and function in all living organisms, and that all cells come from pre-existing cells. [9] Cells emerged on Earth at least 3.5 billion years ago. [10] [11] [12]

Chapter 12 Cell Cycle

1. Explain how cell division functions in reproduction, growth, and repair.

2. Describe the structural organization of a prokaryotic and a eukaryotic genome.

3. Describe the major events of cell division that enable the genome of one cell to be passed on to two daughter cells.

4. Describe how chromosome number changes throughout the human life cycle.

The Mitotic Cell Cycle

5. List the phases of the cell cycle and describe the sequence of events that occurs during each phase.

6. List the phases of mitosis and describe the events characteristic of each phase.

7. Recognize the phases of mitosis from diagrams and micrographs.

8. Draw or describe the spindle apparatus, including centrosomes, kinetochore microtubules, nonkinetochore microtubules, asters, and centrioles (in animal cells).

9. Describe what characteristic changes occur in the spindle apparatus during each phase of mitosis.

10. Explain the current models for poleward chromosomal movement and elongation of the cell’s polar axis.

11. Compare cytokinesis in animals and in plants.

12. Describe the process of binary fission in bacteria and explain how eukaryotic mitosis may have evolved from binary fission.

Regulation of the Cell Cycle

13. Describe the roles of checkpoints, cyclin, Cdk, and MPF in the cell cycle control system.

14. Describe the internal and external factors that influence the cell cycle control system.

15. Explain how the abnormal cell division of cancerous cells escapes normal cell cycle controls.

12.4: Microtubules - Biology

Twister RNAs represent a recently discovered class of natural ribozymes that promote rapid cleaving of RNA backbones. Although an abundance of theoretical, biochemical, and structural data exist for several members of the twister class, disagreements about the architecture and mechanism of its active site have emerged. Historically, such storms regarding mechanistic details typically occur soon after each new self-cleaving ribozyme class is reported, but paths forward exist to quickly reach calmer conditions.

Proteolysis-Targeting Chimeras: Induced Protein Degradation as a Therapeutic Strategy

Until recently, the only ways to reduce specific protein signaling were to either knock down the target by RNAi or to interfere with the signaling by inhibiting an enzyme or receptor within the signal transduction cascade. Herein, we review an emerging class of small molecule pharmacological agents, called PROTACs, that present a novel approach to specifically target proteins and their respective signaling pathways. These heterobifunctional molecules utilize endogenous cellular quality control machinery by recruiting it to target proteins in order to induce their degradation.

Chemoproteomic Screening of Covalent Ligands Reveals UBA5 As a Novel Pancreatic Cancer Target
  • Allison M. Roberts ,
  • David K. Miyamoto ,
  • Tucker R. Huffman ,
  • Leslie A. Bateman ,
  • Ashley N. Ives ,
  • David Akopian ,
  • Martin J. Heslin ,
  • Carlo M. Contreras ,
  • Michael Rape ,
  • Christine F. Skibola , and
  • Daniel K. Nomura*

Chemical genetic screening of small-molecule libraries has been a promising strategy for discovering unique and novel therapeutic compounds. However, identifying the targets of lead molecules that arise from these screens has remained a major bottleneck in understanding the mechanism of action of these compounds. Here, we have coupled the screening of a cysteine-reactive fragment-based covalent ligand library with an isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) chemoproteomic platform to rapidly couple the discovery of lead small molecules that impair pancreatic cancer pathogenicity with the identification of druggable hotspots for potential cancer therapy. Through this coupled approach, we have discovered a covalent ligand DKM 2–93 that impairs pancreatic cancer cell survival and in vivo tumor growth through covalently modifying the catalytic cysteine of the ubiquitin-like modifier activating enzyme 5 (UBA5), thereby inhibiting its activity as a protein that activates the ubiquitin-like protein UFM1 to UFMylate proteins. We show that UBA5 is a novel pancreatic cancer therapeutic target and show DKM 2–93 as a relatively selective lead inhibitor of UBA5. Our results underscore the utility of coupling the screening of covalent ligand libraries with isoTOP-ABPP platforms for mining the proteome for druggable hotspots for cancer therapy.

Argininosuccinate Synthase 1 is a Metabolic Regulator of Colorectal Cancer Pathogenicity
  • Leslie A. Bateman ,
  • Wan-Min Ku ,
  • Martin J. Heslin ,
  • Carlo M. Contreras ,
  • Christine F. Skibola* , and
  • Daniel K. Nomura*

Like many cancer types, colorectal cancers have dysregulated metabolism that promotes their pathogenic features. In this study, we used the activity-based protein profiling chemoproteomic platform to profile cysteine-reactive metabolic enzymes that are upregulated in primary human colorectal tumors. We identified argininosuccinate synthase 1 (ASS1) as an upregulated target in primary human colorectal tumors and show that pharmacological inhibition or genetic ablation of ASS1 impairs colorectal cancer pathogenicity. Using metabolomic profiling, we show that ASS1 inhibition leads to reductions in the levels of oncogenic metabolite fumarate, leading to impairments in glycolytic metabolism that supports colorectal cancer cell pathogenicity. We show here that ASS1 inhibitors may represent a novel therapeutic approach for attenuating colorectal cancer through compromising critical metabolic and metabolite signaling pathways and demonstrate the utility of coupling chemoproteomic and metabolomic strategies to map novel metabolic regulators of cancer.

Characterization of CYP115 As a Gibberellin 3-Oxidase Indicates That Certain Rhizobia Can Produce Bioactive Gibberellin A4

The gibberellin (GA) phytohormones are produced not only by plants but also by fungi and bacteria. Previous characterization of a cytochrome P450 (CYP)-rich GA biosynthetic operon found in many symbiotic, nitrogen-fixing rhizobia led to the elucidation of bacterial GA biosynthesis and implicated GA9 as the final product. However, GA9 does not exhibit hormonal/biological activity and presumably requires further transformation to elicit an effect in the legume host plant. Some rhizobia that contain the GA operon also possess an additional CYP (CYP115), and here we show that this acts as a GA 3-oxidase to produce bioactive GA4 from GA9. This is the first GA 3-oxidase identified for rhizobia, and provides a more complete scheme for biosynthesis of bioactive GAs in bacteria. Furthermore, phylogenetic analyses suggest that rhizobia acquired CYP115 independently of the core GA operon, adding further complexity to the horizontal gene transfer of GA biosynthetic enzymes among bacteria.

Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein
  • Kai Cai ,
  • Ronnie O. Frederick ,
  • Marco Tonelli , and
  • John L. Markley*

Mitochondrial cysteine desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human cysteine desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the cysteine desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.

Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci

Macrocycles represent a common structural framework in many naturally occurring peptides. Several strategies exist for macrocyclization, and the enzymes that incorporate them are of great interest, as they enhance our repertoire for creating complex molecules. We recently discovered a new peptide cyclization reaction involving a crosslink between the side chains of lysine and tryptophan that is installed by a radical SAM enzyme. Herein, we characterize relatives of this metalloenzyme from the pathogens Streptococcus agalactiae and Streptococcus suis. Our results show that the corresponding enzymes, which we call AgaB and SuiB, contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective substrates. Subsequent high-resolution-MS and 2D-NMR analyses located the site of macrocyclization. Moreover, we report that AgaB can accept modified substrates containing natural or unnatural amino acids. Aside from providing insights into the mechanism of this unusual modification, the substrate promiscuity of AgaB may be exploited to create diverse macrocyclic peptides.

A Fluorescent Probe Distinguishes between Inhibition of Early and Late Steps of Lipopolysaccharide Biogenesis in Whole Cells
  • Eileen Moison ,
  • Ran Xie ,
  • Ge Zhang ,
  • Matthew D. Lebar ,
  • Timothy C. Meredith* , and
  • Daniel Kahne*

Lipopolysaccharide (LPS) biogenesis in Gram-negative organisms involves its biosynthesis in the cytoplasm and subsequent transport across three cellular compartments to the cell surface. We developed a fluorescent probe that allows us to determine the spatial distribution of LPS in whole cells. We show that polymyxin B nonapeptide (PMBN) containing a dansyl fluorophore specifically binds to LPS in membranes. We show that this probe detects decreases in LPS levels on the cell surface when LPS biosynthesis is inhibited at an early step. We also can detect accumulation of LPS in particular subcellular locations when LPS assembly is blocked during transport, allowing us to differentiate inhibitors targeting early and late stages of LPS biogenesis.

Structural and Epimeric Isomers of HPPH [3-Devinyl 3-pyropheophorbide-a]: Effects on Uptake and Photodynamic Therapy of Cancer
  • Courtney Saenz ,
  • Ravindra R. Cheruku ,
  • Tymish Y. Ohulchanskyy ,
  • Penny Joshi ,
  • Walter A. Tabaczynski ,
  • Joseph R. Missert ,
  • Yihui Chen ,
  • Paula Pera ,
  • Erin Tracy ,
  • Aimee Marko ,
  • Daniel Rohrbach ,
  • Ulas Sunar ,
  • Heinz Baumann* , and
  • Ravindra K. Pandey*

The tetrapyrrole structure of porphyrins used as photosentizing agents is thought to determine uptake and retention by malignant epithelial cancer cells. To assess the contribution of the oxidized state of individual rings to these cellular processes, bacteriochlorophyll a was converted into the ring “D” reduced 3-devinyl-3-[1-(1-hexyloxy)ethyl]pyropheophorbide-a (HPPH) and the corresponding ring “B” reduced isomer (iso-HPPH). The carboxylic acid analogs of both ring “B” and ring “D” reduced isomers showed several-fold higher accumulation into the mitochondria and endoplasmic reticulum by primary culture of human lung and head and neck cancer cells than the corresponding methyl ester analogs that localize primarily to granular vesicles and to a lesser extent to mitochondria. However, long-term cellular retention of these compounds exhibited an inverse relationship with tumor cells generally retaining better the methyl-ester derivatives. In vivo distribution and tumor uptake was evaluated in the isogenic model of BALB/c mice bearing Colon26 tumors using the respective 14C-labeled analogs. Both carboxylic acid derivatives demonstrated similar intracellular localization and long-term tumor cure with no significant skin phototoxicity. PDT-mediated tumor action involved vascular damage, which was confirmed by a reduction in blood flow and immunohistochemical assessment of damage to the vascular endothelium. The HPPH stereoisomers (epimers) showed identical uptake (in vitro & in vivo), intracellular retention and photoreaction.

Identifying Functional Cysteine Residues in the Mitochondria
  • Daniel W. Bak* ,
  • Mattia D. Pizzagalli , and
  • Eranthie Weerapana*

The mitochondria are dynamic organelles that regulate oxidative metabolism and mediate cellular redox homeostasis. Proteins within the mitochondria are exposed to large fluxes in the surrounding redox environment. In particular, cysteine residues within mitochondrial proteins sense and respond to these redox changes through oxidative modifications of the cysteine thiol group. These oxidative modifications result in a loss in cysteine reactivity, which can be monitored using cysteine-reactive chemical probes and quantitative mass spectrometry (MS). Analysis of cell lysates treated with cysteine-reactive probes enable the identification of hundreds of cysteine residues, however, the mitochondrial proteome is poorly represented (<10% of identified peptides), due to the low abundance of mitochondrial proteins and suppression of mitochondrial peptide MS signals by highly abundant cytosolic peptides. Here, we apply a mitochondrial isolation and purification protocol to substantially increase coverage of the mitochondrial cysteine proteome. Over 1500 cysteine residues from ∼450 mitochondrial proteins were identified, thereby enabling interrogation of an unprecedented number of mitochondrial cysteines. Specifically, these mitochondrial cysteines were ranked by reactivity to identify hyper-reactive cysteines with potential catalytic and regulatory functional roles. Furthermore, analyses of mitochondria exposed to nitrosative stress revealed previously uncharacterized sites of protein S-nitrosation on mitochondrial proteins. Together, the mitochondrial cysteine enrichment strategy presented herein enables detailed characterization of protein modifications that occur within the mitochondria during (patho)physiological fluxes in the redox environment.

The SUV39H1 Protein Lysine Methyltransferase Methylates Chromatin Proteins Involved in Heterochromatin Formation and VDJ Recombination
  • Srikanth Kudithipudi ,
  • Maren Kirstin Schuhmacher ,
  • Adam Fiseha Kebede , and
  • Albert Jeltsch*

SUV39H1 is an H3K9 methyltransferase involved in the formation of heterochromatin. We investigated its substrate specificity profile and show recognition of H3 residues between K4 and G12 with highly specific readout of R8. The specificity profile of SUV39H1 is distinct from its paralog SUV39H2, indicating that they can have different additional substrates. Using the specificity profile, several novel SUV39H1 candidate substrates were identified. We observed methylation of 19 novel substrates at the peptide level and for six of them at the protein level. Methylation of RAG2, SET8, and DOT1L was confirmed in cells, which all have important roles in chromatin regulation. Methylation of SET8 allosterically stimulates its H4K20 monomethylation activity connecting SUV39H1 to the generation of increased H4K20me3 levels, another heterochromatic modification. Methylation of RAG2 alters its subnuclear localization, indicating that SUV39H1 might regulate VDJ recombination. Taken together, our results indicate that beyond the generation of H3K9me3, SUV39H1 has additional roles in chromatin biology by direct stimulation of the establishment of H4K20me3 and the regulation of chromatin binding of RAG2.

Modification of the Orthosteric PPARγ Covalent Antagonist Scaffold Yields an Improved Dual-Site Allosteric Inhibitor
  • Richard Brust ,
  • Hua Lin ,
  • Jakob Fuhrmann ,
  • Alice Asteian ,
  • Theodore M. Kamenecka , and
  • Douglas J. Kojetin*

GW9662 and T0070907 are widely used commercially available irreversible antagonists of peroxisome proliferator-activated receptor gamma (PPARγ). These antagonists covalently modify Cys285 located in an orthosteric ligand-binding pocket embedded in the PPARγ ligand-binding domain and are used to block binding of other ligands. However, we recently identified an alternate/allosteric ligand-binding site in the PPARγ LBD to which ligand binding is not inhibited by these orthosteric covalent antagonists. Here, we developed a series of analogs based on the orthosteric covalent antagonist scaffold with the goal of inhibiting both orthosteric and allosteric cellular activation of PPARγ by MRL20, an orthosteric agonist that also binds to an allosteric site. Our efforts resulted in the identification of SR16832 (compound 22), which functions as a dual-site covalent inhibitor of PPARγ transcription by PPARγ-binding ligands. Molecular modeling, protein NMR spectroscopy structural analysis, and biochemical assays indicate the inhibition of allosteric activation occurs in part through expansion of the 2-chloro-5-nitrobenzamidyl orthosteric covalent antagonist toward the allosteric site, weakening of allosteric ligand binding affinity, and inducing conformational changes not competent for cellular PPARγ activation. Furthermore, SR16832 better inhibits binding of rosiglitazone, a thiazolidinedione (TZD) that weakly activates PPARγ when cotreated with orthosteric covalent antagonists, and may better inhibit binding of endogenous PPARγ ligands such as docosahexaenoic acid (DHA) compared to orthosteric covalent antagonists. Compounds such as SR16832 may be useful chemical tools to use as a dual-site bitopic orthosteric and allosteric covalent inhibitor of ligand binding to PPARγ.

Evolution and Distribution of C7–Cyclitol Synthases in Prokaryotes and Eukaryotes
  • Andrew R. Osborn ,
  • Kelsey M. Kean ,
  • Khaled M. Alseud ,
  • Khaled H. Almabruk ,
  • Shumpei Asamizu ,
  • Janet A. Lee ,
  • P. Andrew Karplus* , and
  • Taifo Mahmud*

2-Epi-5-epi-valiolone synthase (EEVS), a C7-sugar phosphate cyclase (SPC) homologous to 3-dehydroquinate synthase (DHQS), was discovered during studies of the biosynthesis of the C7N-aminocyclitol family of natural products. EEVS was originally thought to be present only in certain actinomycetes, but analyses of genome sequences showed that it is broadly distributed in both prokaryotes and eukaryotes, including vertebrates. Another SPC, desmethyl-4-deoxygadusol synthase (DDGS), was later discovered as being involved in the biosynthesis of mycosporine-like amino acid sunscreen compounds. Current database annotations are quite unreliable, with many EEVSs reported as DHQS, and most DDGSs reported as EEVS, DHQS, or simply hypothetical proteins. Here, we identify sequence features useful for distinguishing these enzymes, report a crystal structure of a representative DDGS showing the high similarity of the EEVS and DDGS enzymes, identify notable active site differences, and demonstrate the importance of two of these active site residues for catalysis by point mutations. Further, we functionally characterized two representatives of a distinct clade equidistant from known EEVS and known DDGS groups and show them to be authentic EEVSs. Moreover, we document and discuss the distribution of genes that encode EEVS and DDGS in various prokaryotes and eukaryotes, including pathogenic bacteria, plant symbionts, nitrogen-fixing bacteria, myxobacteria, cyanobacteria, fungi, stramenopiles, and animals, suggesting their broad potential biological roles in nature.

Determinants of BH3 Sequence Specificity for the Disruption of Bcl-xL/cBid Complexes in Membranes

The prosurvival Bcl-2 proteins exhibit a specific pattern of interactions with BH3-only proteins that determines the cellular dependence on apoptotic stress. This specificity is crucial for the development of BH3 mimetics, a class of anticancer molecules based on the BH3 domain with promising activity in clinical trials. Although complex formation mainly takes place in the mitochondrial outer membrane, most studies so far addressed the interaction between BH3 peptides and truncated Bcl-2 proteins in solution. As a consequence, quantitative understanding of the sequence specificity determinants of BH3 peptides in the membrane environment is missing. Here, we tackle this issue by systematically quantifying the ability of BH3 peptides to compete for the complexes between cBid and Bcl-xL in giant unilamellar vesicles and compare it with solution and mitochondria. We show that the BH3 peptides derived from Hrk, Bim, Bid, and Bad are the most efficient in disrupting cBid/Bcl-xL complexes in the membrane, which correlates with their activity in mitochondria. Our findings support the targeting to the membrane of small molecules that bind Bcl-2 proteins as a strategy to improve their efficiency.

Control of an Unusual Photo-Claisen Rearrangement in Coumarin Caged Tamoxifen through an Extended Spacer
  • Pamela T. Wong ,
  • Edward W. Roberts ,
  • Shengzhuang Tang ,
  • Jhindan Mukherjee ,
  • Jayme Cannon ,
  • Alyssa J. Nip ,
  • Kaitlin Corbin ,
  • Matthew F. Krummel* , and
  • Seok Ki Choi*

The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.

The Activity of JmjC Histone Lysine Demethylase KDM4A is Highly Sensitive to Oxygen Concentrations
  • Rebecca L Hancock ,
  • Norma Masson ,
  • Kate Dunne ,
  • Emily Flashman* , and
  • Akane Kawamura*

The JmjC histone lysine demethylases (KDMs) are epigenetic regulators involved in the removal of methyl groups from post-translationally modified lysyl residues within histone tails, modulating gene transcription. These enzymes require molecular oxygen for catalytic activity and, as 2-oxoglutarate (2OG)-dependent oxygenases, are related to the cellular oxygen sensing HIF hydroxylases PHD2 and FIH. Recent studies have indicated that the activity of some KDMs, including the pseudogene-encoded KDM4E, may be sensitive to changing oxygen concentrations. Here, we report detailed analysis of the effect of oxygen availability on the activity of the KDM4 subfamily member KDM4A, importantly demonstrating a high level of O2 sensitivity both with isolated protein and in cells. Kinetic analysis of the recombinant enzyme revealed a high KMapp(O2) of 173 ± 23 μM, indicating that the activity of the enzyme is able to respond sensitively to a reduction in oxygen concentration. Furthermore, immunofluorescence experiments in U2OS cells conditionally overexpressing KDM4A showed that the cellular activity of KDM4A against its primary substrate, H3K9me3, displayed a graded response to depleting oxygen concentrations in line with the data obtained using isolated protein. These results suggest that KDM4A possesses the potential to act as an oxygen sensor in the context of chromatin modifications, with possible implications for epigenetic regulation in hypoxic disease states. Importantly, this correlation between the oxygen sensitivity of the catalytic activity of KDM4A in biochemical and cellular assays demonstrates the utility of biochemical studies in understanding the factors contributing to the diverse biological functions and varied activity of the 2OG oxygenases.

O-GlcNAcylation of α-Synuclein at Serine 87 Reduces Aggregation without Affecting Membrane Binding
  • Yuka E. Lewis ,
  • Ana Galesic ,
  • Paul M. Levine ,
  • Cesar A. De Leon ,
  • Natalie Lamiri ,
  • Caroline K. Brennan , and
  • Matthew R. Pratt*

The aggregation of neurodegenerative-disease associated proteins can be affected by many factors, including a variety of post-translational modifications. One such modification, O-GlcNAcylation, has been found on some of these aggregation prone proteins, including α-synuclein, the major protein that plays a causative role in synucleinopathies like Parkinson’s disease. We previously used synthetic protein chemistry to prepare α-synuclein bearing a homogeneous O-GlcNAc modification at threonine 72 and showed that this modification inhibits protein aggregation. However, the effects of the other eight O-GlcNAcylation sites that have been identified were unknown. Here, we use a similar synthetic strategy to investigate the consequences of this modification at one of these sites, serine 87. We show that O-GlcNAcylation at this site also inhibits α-synuclein aggregation but to a lesser extent than that for the same modification at threonine 72. However, we also find that this modification does not affect the membrane-binding properties of α-synuclein, which differentiates it from phosphorylation at the same site. These results further support the development of therapies that can elevate O-GlcNAcylation of α-synuclein to slow the progression of Parkinson’s disease.

Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors
  • Joel R. Walker* ,
  • Mary P. Hall ,
  • Chad A. Zimprich ,
  • Matthew B. Robers ,
  • Sarah J. Duellman ,
  • Thomas Machleidt ,
  • Jacquelynn Rodriguez , and
  • Wenhui Zhou

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.

Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity
  • Geng-Yuan Chen ,
  • You Jung Kang ,
  • A. Sophia Gayek ,
  • Wiphu Youyen ,
  • Erkan Tüzel ,
  • Ryoma Ohi , and
  • William O. Hancock*

To uncover their contrasting mechanisms, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 “braking” dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM KI. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.

A Fluorescent Hsp90 Probe Demonstrates the Unique Association between Extracellular Hsp90 and Malignancy in Vivo
  • Lauren B. Crowe ,
  • Philip F. Hughes ,
  • David A. Alcorta ,
  • Takuya Osada ,
  • Aaron P. Smith ,
  • Juliane Totzke ,
  • David R. Loiselle ,
  • Isaac D. Lutz ,
  • Madhusudhana Gargesha ,
  • Debasish Roy ,
  • Jose Roques ,
  • David Darr ,
  • H. Kim Lyerly ,
  • Neil L. Spector , and
  • Timothy A.J. Haystead*

Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to target eHsp90 in vivo. High resolution confocal and lattice light sheet microscopy show that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is actively internalized. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by overexpressing p110HER2. Whole body cryoslicing, imaging, and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression and internalization is a phenomenon unique to tumor cells in vivo and may provide an “Achilles heel” for the early diagnosis of metastatic disease and targeted drug delivery.

Iron Release from the Siderophore Pyoverdine in Pseudomonas aeruginosa Involves Three New Actors: FpvC, FpvG, and FpvH
  • Géraldine Ganne ,
  • Karl Brillet ,
  • Beata Basta ,
  • Béatrice Roche ,
  • Françoise Hoegy ,
  • Véronique Gasser , and
  • Isabelle J. Schalk*

Siderophores are iron chelators produced by bacteria to access iron, an essential nutriment. Pyoverdine (PVDI), the major siderophore produced by Pseudomonas aeruginosa PAO1, consists of a fluorescent chromophore linked to an octapeptide. The ferric form of PVDI is transported from the extracellular environment into the periplasm by the outer membrane transporter, FpvA. Iron is then released from the siderophore in the periplasm by a mechanism that does not involve chemical modification of the chelator but an iron reduction step. Here, we followed the kinetics of iron release from PVDI, in vitro and in living cells, by monitoring its fluorescence (as apo PVDI is fluorescent, whereas PVDI-Fe(III) is not). Deletion of the inner membrane proteins fpvG (PA2403) and fpvH (PA2404) affected 55Fe uptake via PVDI and completely abolished PVDI-Fe dissociation, indicating that these two proteins are involved in iron acquisition via this siderophore. PVDI-Fe dissociation studies, using an in vitro assay, showed that iron release from this siderophore requires the presence of an iron reducer (DTT) and an iron chelator (ferrozine). In this assay, DTT could be replaced by the inner membrane protein, FpvG, and ferrozine by the periplasmic protein, FpvC, suggesting that FpvG acts as a reductase and FpvC as an Fe2+ chelator in the process of PVDI-Fe dissociation in the periplasm of P. aeruginosa cells. This mechanism of iron release from PVDI is atypical among Gram-negative bacteria but seems to be conserved among Pseudomonads.

The GCaMP-R Family of Genetically Encoded Ratiometric Calcium Indicators
  • Jung-Hwa Cho ,
  • Carter J. Swanson ,
  • Jeannie Chen ,
  • Ang Li ,
  • Lisa G. Lippert ,
  • Shannon E. Boye ,
  • Kasey Rose ,
  • Sivaraj Sivaramakrishnan ,
  • Cheng-Ming Chuong , and
  • Robert H. Chow*

We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins. The tandem design assures that the two proteins localize in the same cellular compartment(s) and facilitates pixelwise ratiometric measurements however, Förster resonance energy transfer (FRET) between the fluorophores reduces brightness of the sensor by up to half (depending on the GCaMP variant). To eliminate FRET, we introduced a rigid α-helix, the ER/K helix, between GCaMP and mCherry. Avoiding FRET significantly increases the brightness (notably, even at low calcium concentrations), the signal-to-noise ratio, and the dynamic range.

Discovery and Characterization of a Potent and Specific Peptide Ligand Targeting Endothelial Progenitor Cells and Endothelial Cells for Tissue Regeneration
  • Dake Hao ,
  • Wenwu Xiao ,
  • Ruiwu Liu ,
  • Priyadarsini Kumar ,
  • Yuanpei Li ,
  • Ping Zhou ,
  • Fuzheng Guo ,
  • Diana L. Farmer ,
  • Kit S. Lam ,
  • Fengshan Wang* , and
  • Aijun Wang*

Endothelial progenitor cells (EPCs) and endothelial cells (ECs) play a vital role in endothelialization and vascularization for tissue regeneration. Various EPC/EC targeting biomolecules have been investigated to improve tissue regeneration with limited success often due to their limited functional specificity and structural stability. One-bead one-compound (OBOC) combinatorial technology is an ultrahigh throughput chemical library synthesis and screening method suitable for ligand discovery against a wide range of biological targets, such as integrins. In this study, using primary human EPCs/ECs as living probes, we identified an αvβ3 integrin ligand LXW7 discovered by OBOC combinatorial technology as a potent and specific EPC/EC targeting ligand. LXW7 overcomes the major barriers of other functional biomolecules that have previously been used to improve vascularization for tissue regeneration and possesses optimal stability, EPC/EC specificity, and functionality. LXW7 is a disulfide cyclic octa-peptide (cGRGDdvc) containing unnatural amino acids flanking both sides of the main functional motif therefore it will be more resistant to proteolysis and more stable in vivo compared to linear peptides and peptides consisting of only natural amino acids. Compared with the conventional αvβ3 integrin ligand GRGD peptide, LXW7 showed stronger binding affinity to primary EPCs/ECs but weaker binding to platelets and no binding to THP-1 monocytes. In addition, ECs bound to the LXW7 treated culture surface exhibited enhanced biological functions such as proliferation, likely due to increased phosphorylation of VEGF receptor 2 (VEGF-R2) and activation of mitogen-activated protein kinase (MAPK) ERK1/2. Surface modification of electrospun microfibrous PLLA/PCL biomaterial scaffolds with LXW7 via Click chemistry resulted in significantly improved endothelial coverage. LXW7 and its derivatives hold great promise for EPC/EC recruitment and delivery and can be widely applied to functionalize various biological and medical materials to improve endothelialization and vascularization for tissue regeneration applications.

Fluorescent Hexose Conjugates Establish Stringent Stereochemical Requirement by GLUT5 for Recognition and Transport of Monosaccharides
  • Olivier-Mohamad Soueidan ,
  • Thomas W. Scully ,
  • Jatinder Kaur ,
  • Rashmi Panigrahi ,
  • Alexandr Belovodskiy ,
  • Victor Do ,
  • Carson D. Matier ,
  • M. Joanne Lemieux ,
  • Frank Wuest ,
  • Chris Cheeseman* , and
  • F. G. West*

The specificity characteristics of transporters can be exploited for the development of novel diagnostic therapeutic probes. The facilitated hexose transporter family (GLUTs) has a distinct set of preferences for monosaccharide substrates, and while some are expressed ubiquitously (e.g., GLUT1), others are quite tissue specific (e.g., GLUT5, which is overexpressed in some breast cancer tissues). While these differences have enabled the development of new molecular probes based upon hexose- and tissue-selective uptake, substrate design for compounds targeting these GLUT transporters has been encumbered by a limited understanding of the molecular interactions at play in hexose binding and transport. Four new fluorescently labeled hexose derivatives have been prepared, and their transport characteristics were examined in two breast cancer cell lines expressing mainly GLUTs 1, 2, and 5. Our results demonstrate, for the first time, a stringent stereochemical requirement for recognition and transport by GLUT5. 6-NBDF, in which all substituents are in the d-fructose configuration, is taken up rapidly into both cell lines via GLUT5. On the other hand, inversion of a single stereocenter at C-3 (6-NBDP), C-4 (6-NBDT), or C-5 (6-NDBS) results in selective transport via GLUT1. An in silico docking study employing the recently published GLUT5 crystal structure confirms this stereochemical dependence. This work provides insight into hexose-GLUT interactions at the molecular level and will facilitate structure-based design of novel substrates targeting individual members of the GLUT family and forms the basis of new cancer imaging or therapeutic agents.

The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase
  • Elise A. Span ,
  • Daniel L. M. Suess ,
  • Marc C. Deller ,
  • R. David Britt , and
  • Michael A. Marletta*

Polysaccharide monooxygenases (PMOs) are secreted metalloenzymes that catalyze the oxidative degradation of polysaccharides in a copper-, oxygen-, and reductant-dependent manner. Cellulose-active fungal PMOs degrade cellulosic substrates to be utilized as a carbon source for fungal growth. To gain insight into the PMO mechanism, the role of conserved residues in the copper coordination sphere was investigated. Here, we report active-site hydrogen-bonding motifs in the secondary copper coordination sphere of MtPMO3*, a C1-oxidizing PMO from the ascomycete fungus Myceliophthora thermophila. A series of point substitutions that disrupt this conserved network are used to interrogate its function. Activity assays, in conjunction with EPR spectroscopy, demonstrate that residues H161 and Q167 are involved in stabilizing bound oxygen, and H161 appears to play a role in proton transfer. Additionally, Q167 increases the ligand donor strength of Y169 to the copper via a hydrogen-bonding interaction. Altogether, H161 and Q167 are important for oxygen activation, and the results are suggestive of a copper–oxyl active intermediate.

NMR and Molecular Recognition of N-Glycans: Remote Modifications of the Saccharide Chain Modulate Binding Features
  • Ana Gimeno ,
  • Niels-Christian Reichardt ,
  • F. Javier Cañada ,
  • Lukas Perkams ,
  • Carlo Unverzagt ,
  • Jesús Jiménez-Barbero* , and
  • Ana Ardá*

Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate–protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore their importance for the development of biomedical applications.

Ion Mobility-Mass Spectrometry Reveals a Dipeptide That Acts as a Molecular Chaperone for Amyloid β
  • Molly T. Soper-Hopper ,
  • Joseph D. Eschweiler , and
  • Brandon T. Ruotolo*

Previously, we discovered and structurally characterized a complex between amyloid β 1–40 and the neuropeptide leucine enkephalin. This work identified leucine enkephalin as a potentially useful starting point for the discovery of peptide-related biotherapeutics for Alzheimer’s disease. In order to better understand such complexes that are formed in vitro, we describe here the analysis of a series of site-directed amino acid substitution variants of both peptides, covering the leucine enkephalin sequence in its entirety and a large number of selected residues of amyloid β 1–40 (residues: D1, E3, F4, R5, H6, Y10, E11, H13, H14, Q15, K16, E22, K28, and V40). Ion mobility–mass spectrometry measurements and molecular dynamics simulations reveal that the hydrophobic C-terminus of leucine enkephalin (Phe-Leu, FL) is crucial for the formation of peptide complexes. As such, we explore here the interaction of the dipeptide FL with both wildtype and variant forms of amyloid β in order to structurally characterize the complexes formed. We find that FL binds preferentially to amyloid β oligomers and attaches to amyloid β within the region between its N-terminus and its hydrophobic core, most specifically at residues Y10 and Q15. We further show that FL is able to prevent fibril formation.

A Near-Infrared, Wavelength-Shiftable, Turn-on Fluorescent Probe for the Detection and Imaging of Cancer Tumor Cells
  • Zhenhua Shen ,
  • Bijeta Prasai ,
  • Yuko Nakamura ,
  • Hisataka Kobayashi ,
  • Milcah S. Jackson , and
  • Robin L. McCarley*

Fast, selective, and noninvasive reporting of intracellular cancer-associated events and species will lead to a better understanding of tumorigenesis at the molecular level and development of precision medicine approaches in oncology. Overexpressed reductase presence in solid tumor cells is key to cancer progression and protection of those diseased cells from the oxidative effects of therapeutics meant to kill them. Human NAD(P)H:quinone oxidoreductase isozyme I (hNQO1), a cytoprotective 2-electron-specific reductase found at unusually high activity levels in cancer cells of multiple origins, has attracted significant attention due to its major role in metastatic pathways and its link to low survival rates in patients, as well as its ability to effectively activate quinone-based, anticancer drugs. Accurate assessment of hNQO1 activities in living tumor models and ready differentiation of metastases from healthy tissue by fluorescent light-based protocols requires creation of hNQO1-responsive, near-infrared probes that offer deep tissue penetration and low background fluorescence. Herein, we disclose a quinone-trigger-based, near-infrared probe whose fluorescence is effectively turned on several hundred-fold through highly selective reduction of the quinone trigger group by hNQO1, with unprecedented, catalytically efficient formation of a fluorescent reporter. hNQO1 activity-specific production of a fluorescence signal in two-dimensional cultures of respiring human cancer cells that harbor the reductase enzyme allows for their quick (30 min) high-integrity recognition. The characteristics of the near-infrared probe make possible the imaging of clinically relevant three-dimensional colorectal tumor models possessing spatially heterogeneous hNQO1 activities and provide for fluorescence-assisted identification of submillimeter dimension metastases in a preclinical mouse model of human ovarian serous adenocarcinoma.

Mapping Novel Metabolic Nodes Targeted by Anti-Cancer Drugs that Impair Triple-Negative Breast Cancer Pathogenicity
  • Lindsay S. Roberts ,
  • Peter Yan ,
  • Leslie A. Bateman , and
  • Daniel K. Nomura*

Triple-negative breast cancers (TNBCs) are estrogen receptor, progesterone receptor, and HER2 receptor-negative subtypes of breast cancers that show the worst prognoses and lack targeted therapies. Here, we have coupled the screening of ∼400 anticancer agents that are under development or in the clinic with chemoproteomic and metabolomic profiling to identify novel metabolic mechanisms for agents that impair TNBC pathogenicity. We identify 20 anticancer compounds that significantly impaired cell survival across multiple types of TNBC cells. Among these 20 leads, the phytoestrogenic natural product licochalcone A was of interest, since TNBCs are unresponsive to estrogenic therapies, indicating that licochalcone A was likely acting through another target. Using chemoproteomic profiling approaches, we reveal that licochalcone A impairs TNBC pathogenicity, not through modulating estrogen receptor activity but rather through inhibiting prostaglandin reductase 1, a metabolic enzyme involved in leukotriene B4 inactivation. We also more broadly performed metabolomic profiling to map additional metabolic mechanisms of compounds that impair TNBC pathogenicity. Overlaying lipidomic profiling with drug responses, we find that deubiquitinase inhibitors cause dramatic elevations in acyl carnitine levels, which impair mitochondrial respiration and contribute to TNBC pathogenic impairments. We thus put forth two unique metabolic nodes that are targeted by drugs or drug candidates that impair TNBC pathogenicity. Our results also showcase the utility of coupling drug screens with chemoproteomic and metabolomic profiling to uncover unique metabolic drivers of TNBC pathogenicity.

Structure–Activity Relationships of the Competence Stimulating Peptides (CSPs) in Streptococcus pneumoniae Reveal Motifs Critical for Intra-group and Cross-group ComD Receptor Activation
  • Yifang Yang ,
  • Bimal Koirala ,
  • Lucia A. Sanchez ,
  • Naiya R. Phillips ,
  • Sally R. Hamry , and
  • Yftah Tal-Gan*

Streptococcus pneumoniae is a highly recombinogenic human pathogen that utilizes the competence stimulating peptide (CSP)-based quorum sensing (QS) circuitry to acquire antibiotic resistance genes from the environment and initiate its attack on the human host. Modulation of QS in this bacterium, either inhibition or activation, can therefore be used to attenuate S. pneumoniae infectivity and slow down pneumococcal resistance development. In this study, we set to determine the molecular mechanism that drives CSP:receptor binding and identify CSP-based QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in the two major CSP signals (CSP1 and CSP2) and assessed the ability of the mutated analogs to modulate QS against both cognate and noncognate ComD receptors. We then evaluated the overall 3D structures of these analogs using circular dichroism (CD) to correlate between the structure and function of these peptides. Our CD analysis revealed a strong correlation between α-helicity and bioactivity for both specificity groups (CSP1 and CSP2). Furthermore, we identified the first pan-group QS activator and the most potent group-II QS inhibitor to date. These chemical probes can be used to study the role of QS in S. pneumoniae and as scaffolds for the design of QS-based anti-infective therapeutics against S. pneumoniae infections.

Glycation of Lysozyme by Glycolaldehyde Provides New Mechanistic Insights in Diabetes-Related Protein Aggregation
  • Laura Mariño ,
  • Carlos Andrés Maya-Aguirre ,
  • Kris Pauwels ,
  • Bartolomé Vilanova ,
  • Joaquin Ortega-Castro ,
  • Juan Frau ,
  • Josefa Donoso , and
  • Miquel Adrover*

Glycation occurs in vivo as a result of the nonenzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA, or lipids. Protein glycation causes loss-of-function and, consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question of whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the micromolar range). At low HEWL concentration, non-cross-linking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have little impact on HEWL surface hydrophobicity and, therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shifts toward the formation of intermolecular cross-links, which triggers a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic interactions. Additionally, their comparison constitutes the first experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.

Carbocyanine Dyes

Short-Chain Carbocyanine Dyes

Terasaki and co-workers used the short-chain carbocyanine DiOC6(3) (D273) to visualize the ER in both live and aldehyde-fixed cells. This dye and the similar DiOC5(3) have since been used extensively to study structural interactions and dynamics of the ER in neurons, yeast and onion epidermis, and to examine the morphological relationships between the ER, mitochondria, intermediate filaments and microtubules in various cell types. DiOC6(3) and DiOC5(3) pass through the plasma membrane and stain intracellular membranes with a fluorescein-like fluorescence ER membranes can easily be distinguished by their characteristic morphology. Caution must be exercised, however, in using the carbocyanines as probes for the ER. It has been reported that ER staining with DiOC6(3) does not occur until the mitochondria round up and lose the fluorochrome. Rhodamine 6G and the hexyl ester of rhodamine B (R634, R648MP Probes for Mitochondria—Section 12.2) appear to stain like DiOC6(3), except they are apparently less toxic and they fluoresce orange, providing possibilities for multicolor labeling. When used at very low concentrations, these slightly lipophilic rhodamine dyes tend to stain only mitochondria of live cells.

Long-Chain Carbocyanine Dyes

Terasaki and Jaffe have used the long-chain carbocyanines DiIC16(3) and DiIC18(3) (D384, D282) to label ER membranes. They achieved selective labeling of the ER by microinjecting a saturated solution of DiI in oil into sea urchin eggs. This method has been successful in several other egg types but was not effective in molluscan or arthropod axons. As noted in the discussion of dialkylcarbocyanine and dialkylaminostyryl probes in Dialkylcarbocyanine and Dialkylaminostyryl Probes—Section 13.4, DiI diffuses only in continuous membranes.


Important advances in our understanding of the cytoskeleton have been made by direct observations of living cells following microinjection with fluorescent derivatives of cytoskeletal proteins (Desai and Mitchison, 1997). More recently, however, the ability to express cloned proteins containing a green fluorescent protein (GFP) tag has become the method of choice for dynamic analysis of the cytoskeleton (Chalfie et al., 1994). GFP technology offers several significant advantages over the previous technology: it is not necessary to microinject cells, nor is it necessary to biochemically purify and fluorescently modify the protein of interest. However, there are also limitations to GFP technology: addition of GFP (238 amino acids) to the C or N terminus of the target protein can potentially interfere with protein function. In this regard, it is important to demonstrate that the chimeric protein retains its normal characteristics. In addition, overexpression of any protein can potentially interfere with cellular functions. This latter limitation can be overcome by the use of inducible promoters in the plasmid construct or by establishing permanent cell lines with the desired level of expression of the chimera.

To date, the dynamics of several cytoskeletal proteins have been examined using GFP technology. For example, transformation of yeast with a GFP-actin construct was used to document the motion of cortical actin patches (Doyle and Botstein, 1996). Although the GFP-actin did incorporate into dynamic actin-containing structures in the cells, the construct was not able to complement an actin null mutant (Doyle and Botstein, 1996). The major yeast tubulin gene tub1 has also been tagged with GFP and this construct rescues a tub1mutant (Straight et al., 1997). Importantly, observation of mitosis in yeast transformed with GFP-tub1 provides strong evidence that spindle microtubules can undergo normal dynamic behavior in the expressing cells (Straight et al., 1997). In other experiments, a fusion of GFP to the amino terminus of Tub1p did not complement a tub1 deletion mutation, but yeast cells expressing a mixture of GFP-tagged and wild-type tubulin grew at normal rates (Maddox et al., 1999). The dynamic behavior of individual microtubules has also been examined in yeast expressing an amino terminal fusion of GFP to a different yeast tubulin gene,tub3. The results show that yeast microtubules undergo dynamic instability behavior that is cell cycle regulated (Carminati and Stearns, 1997 Tirnauer et al., 1999). In these experiments, addition of GFP to the amino terminus of tub3, but not to the carboxy terminus, was able to complement atub3 null mutation. Thus, the available data strongly support the view that expression of GFP-tubulin and its incorporation into microtubules does not detectably interfere with microtubule functions in yeast, and is therefore a valuable probe for analysis of microtubule behavior.

Heretofore, it has been extremely difficult to directly measure microtubule dynamics in mammalian cells throughout the cell cycle because of the difficulty of coordinating microinjection of fluorescent tubulin with the cell cycle and the fact that mitotic cells represent only a small fraction of the cells in a population. Other methods to visualize individual microtubules, such as differential interference contrast microscopy, are also more difficult in mitotic cells given their generally rounded morphology (Hayden et al., 1990). Cells expressing GFP-tubulin have the potential to be an invaluable tool for studying microtubule dynamics, organization, and behavior throughout the cell cycle. To date, transient expression of GFP-tagged mouse β6-tubulin in cultured cells strongly suggests that microtubule dynamic behavior is not altered by expression of the GFP construct, although quantitative analysis of microtubule dynamics in these cells was not performed (Ludin and Matus, 1998 Heidemann et al., 1999). In this work, we demonstrate that a cell line permanently expressing GFP-tubulin can be prepared and that the dynamic behavior of interphase microtubules in these cells is very similar to that in parental cells injected with rhodamine-labeled tubulin. We have used these cells to directly measure the changes in microtubule behavior throughout the cell cycle. In contrast to previous results inXenopus egg extracts (Belmont et al., 1990 Verdeet al., 1992 Tournebize et al., 2000), our results demonstrate that both the frequency of catastrophe and of rescue are altered in mitotic compared with interphase cells. The percentage of time microtubules spend in an attenuated state, or paused, is also dramatically reduced in mitotic cells. The rates of elongation and rapid shortening are not changed. In addition to quantification of microtubule dynamic instability in mitotic cells, we document microtubule release from the centrosome and microtubule tethering at the cell cortex. The availability of cells expressing GFP-tubulin should provide a simple, easily manipulated system to examine microtubule behavior in mammalian cells.

12.4: Microtubules - Biology

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Microtubules the thickest cytoskeletal elements in cells are hollow structures that consist of paired globular proteins, alpha and beta tubulins.

These heterodimers form linear rows called protofilaments, which have structural polarity. Meaning that each array is arranged with plus and minus ends. On the plus end where beta tubulins are exposed dimers are added. In contract, on the minus side where alpha tubulins are outward facing dissociation occurs.

However, in other cases microtubules secure stability by directly binding with different proteins like microtubule-associated proteins.

In addition their polarity allows for directional movement throughout the cytoplasm as is the case with dynein and kinesin motor proteins that efficiently transport various cargoes like vesicles.

Microtubules are also key components of cilia and flagella which are specialized extensions that move fluid over the surface of stationary cells and function as propellers in other cells moving them throughout their environments.

In the end, whether they're involved in chromosomal separation during cell division, transporting vesicles in the brain, or sweeping debris out of the lungs microtubules are essential for the growth and development, organizational strength and support, and motility that cells need.

4.10: Microtubules

There are three types of cytoskeletal structures in eukaryotic cells&mdashmicrofilaments, intermediate filaments, and microtubules. With a diameter of about 25 nm, microtubules are the thickest of these fibers. Microtubules carry out a variety of functions that include cell structure and support, transport of organelles, cell motility (movement), and the separation of chromosomes during cell division.

Microtubules are hollow tubes whose walls are made up of globular tubulin proteins. Each tubulin molecule is a heterodimer, consisting of a subunit of &alpha-tubulin and a subunit of &beta-tubulin. The dimers are arranged in linear rows called protofilaments. A microtubule usually consists of 13 protofilaments, arranged side by side, wrapped around the hollow core.

Because of this arrangement, microtubules are polar, meaning that they have different ends. The plus end has &beta-tubulin exposed, and the minus end has &alpha-tubulin exposed. Microtubules can rapidly assemble&mdashgrow in length through polymerization of tubulin molecules&mdashand disassemble. The two ends behave differently in this regard. The plus end is typically the fast-growing end or the end where tubulin is added, and the minus end is the slow-growing end or the end where tubulin dissociates&mdashdepending on the situation.

This process of dynamic instability, where microtubules rapidly grow and shrink, is important for functions such as the remodeling of the cytoskeleton during cell division and the extension of axons from growing neurons.

Microtubules also can be stable, often by binding to microtubule-associated proteins, which help the cell to maintain its shape. Other proteins, called motor proteins, can interact with microtubules to transport organelles in a particular direction. For example, many neurotransmitters are packaged into vesicles in the cell body of a neuron and are then transported down the axon along a &ldquotrack&rdquo of microtubules, delivering the vesicles to where they are needed. Finally, microtubules can also protrude outside of the cell&mdashmaking up the filamentous flagella and cilia that move to push cells (such as sperm) along, or to move fluid across their surfaces, such as in the lungs.

Brouhard, Gary J., and Luke M. Rice. &ldquoMicrotubule Dynamics: An Interplay of Biochemistry and Mechanics.&rdquo Nature Reviews. Molecular Cell Biology 19, no. 7 (July 2018): 451&ndash63. [Source]

Hashimoto, Takashi. &ldquoMicrotubules in Plants.&rdquo The Arabidopsis Book / American Society of Plant Biologists 13 (April 27, 2015). [Source]

Weekly Reflection (12/4-8)

This diagram is of the phospholipid bilayer and how different parts of the cell interact with each other.

During this week, we focused primarily on cell structure and how different parts of the cell work with each other. We also did an in class worksheet on cell parts and watched a video about the interior of a white blood cell (leukocyte). We lectured on cell structure as well which I found helpful.

The cell membrane is made up of a phospholipid bilayer and proteins and is an important part in keeping the shape of a cell. component in maintaining the shape of a cell. This part of cell helps to create a “boundary” which helps to monitor what is being let into and out of the cell through the bilayer. Only certain organic material that is able to pass through the semipermeable membrane can enter the cell. Excess or unwanted materials can be pushed out of the cell through the lipidbilayer if the cell has no use for it anymore. The (phospholipid)bilayer creates fluidity because of the constant movemenet of the phopholipids (heads and tails). Another key component of the membrane is cholesterol molecules. They are in charge of acting as a temperature buffer to maintain the fluidity of the cell. Both integral and peripheral proteins can be found on the membrane and are responsible for letting different materials in and out of the cell. The difference between intergal proteins penetrate the bilayer and while peripheral proteins on the other hand do not penetrate the bilayer. Both help with cell to cell recognition, transportation, signal transduction, and varies enzymatic activity. Another component to the cell membrane is signal transduction which is the receipt of chemical messages from the environment and the relay of those messages into the cell for response. An example of this is how animal cells rely on cell mebranes, but other cells have cell walls. The other type of cell, plant cells, have walls are made up of cellulose, fungal cell walls are made up of chitin, and bacterial cell walls are made up of peptidoglycan. That is the crucial difference between both animal and plant cells.

This diagram shows the phospholipid bilayer and where the integral and peripheral proteins lie in the bilayer.

Another part of the cell that we talked about this week was the cytoskeleton. The Cytoskeleton is a network of structural proteins that extends throughout the cytoplasm. It’s made up of microtubles, actin filaments, and intermediate filaments. Microtubules are in charge of moving organelles, with help of motor proteins (transport proteins), throughout the cell. A way to think about Mircortubules is like a cable car track that helps to move material to different parts of the cell. The centrosome is special because it is only found in animal cells. It is where microtubules originate from. The last part of the cytoskeleton are the microfilaments. They are responsible for the changes in a cells shape which important with interactions with other cells. The intermediate filaments help to prevent tension and ground the nucleus. All parts of the cytoskeleton help to make up the structure of each and every cell in your body and helps to maintain consistency.

This picture is of the cytoskeleton. It shows the filaments, mitochondria, and microtubules.

The last part of the cell that we learned about this week was extracellular matrix (ECM) which is absolutely critical to a cell. The ECM is a network of connective proteins and protoglycan molecules outside of the cell membrane that help with cell anchorage and cell communication. The intercellular junction is a type of protein that helps to connect cells to other cells. The other type of protein that is part of the ECM are open junctions. They allow hydrophilic molecules or ions to pass through from cell to cell. They also help to to “glue” the cells together (helps with cell shape) and creates a waterproof seal between the multiple cells.

This past week, we talked about the ideas that connect to Big idea 3 and Big idea 4. Big idea 3 focuses on cell communication and transmission f signals (think signal transduction). Big idea 4 is how special molecules differentiate from each other.

Dilution-induced disassembly of microtubules: Relation to dynamic instability and the GTP cap

Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15-fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubules of 60 μm/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107:1437–1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP-tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.

12.4: Microtubules - Biology

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Feature Papers represent the most advanced research with significant potential for high impact in the field. Feature Papers are submitted upon individual invitation or recommendation by the scientific editors and undergo peer review prior to publication.

The Feature Paper can be either an original research article, a substantial novel research study that often involves several techniques or approaches, or a comprehensive review paper with concise and precise updates on the latest progress in the field that systematically reviews the most exciting advances in scientific literature. This type of paper provides an outlook on future directions of research or possible applications.

Editor’s Choice articles are based on recommendations by the scientific editors of MDPI journals from around the world. Editors select a small number of articles recently published in the journal that they believe will be particularly interesting to authors, or important in this field. The aim is to provide a snapshot of some of the most exciting work published in the various research areas of the journal.