PM534, an Optimized Target-Protein Interaction Strategy through the Colchicine Site of Tubulin.

Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure-Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the , family , genus (du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model.

Chemical modulation of microtubule structure through the laulimalide/peloruside site.

Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics.

Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function.

Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds.

Crystal Structure of the Cyclostreptin-Tubulin Adduct: Implications for Tubulin Activation by Taxane-Site Ligands.

It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to βHis229, but no stabilization of the M-loop.

Analyzing Mitotic Chromosome Structural Defects After Topoisomerase II Inhibition or Mutation.

For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction we have adapted classical cell cycle experiments, classical cytological techniques and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the protocols used and we discuss the rational for our choice and for the adaptations applied. We clarify in which cell cycle stages each of the different chromosomal aberrations induced by inhibiting topo II takes place: lack of chromosome segregation, undercondensation, lack of sister chromatid resolution, and lack of chromosome individualization.

RepA-WH1 prionoid: Clues from bacteria on factors governing phase transitions in amyloidogenesis.

In bacterial plasmids, Rep proteins initiate DNA replication by undergoing a structural transformation coupled to dimer dissociation. Amyloidogenesis of the 'winged-helix' N-terminal domain of RepA (WH1) is triggered in vitro upon binding to plasmid-specific DNA sequences, and occurs at the bacterial nucleoid in vivo. Amyloid fibers are made of distorted RepA-WH1 monomers that assemble as single or double intertwined tubular protofilaments.

A novel chromatin tether domain controls topoisomerase IIα dynamics and mitotic chromosome formation.

DNA topoisomerase IIα (Topo IIα) is the target of an important class of anticancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA. We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes.

Cohesin is needed for bipolar mitosis in human cells.

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells.

Determinants of Rad21 localization at the centrosome in human cells.

Cohesin proteins help maintain the physical associations between sister chromatids that arise in S-phase and are removed in anaphase. Recent studies found that cohesins also localize to the centrosomes, the organelles that organize the mitotic bipolar spindle. We find that the cohesin protein Rad21 localizes to centrosomes in a manner that is dependent upon known regulators of sister chromatid cohesion as well as regulators of centrosome function.

Rad21 is required for centrosome integrity in human cells independently of its role in chromosome cohesion.

Classically, chromosomal functions in DNA repair and sister chromatid association have been assigned to the cohesin proteins. More recent studies have provided evidence that cohesins also localize to the centrosomes, which organize the bipolar spindle during mitosis. Depletion of cohesin proteins is associated with multi-polar mitosis in which spindle pole integrity is compromised.

Cytological analysis of chromosome structural defects that result from topoisomerase II dysfunction.

For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction, we have adapted classical cell cycle experiments, classical cytological techniques, and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the protocols used and we discuss the rationale for our choice and for the adaptations applied. We clarify in which cell cycle stages each of the different chromosomal aberrations induced by inhibiting topo II take place: lack of chromosome segregation, undercondensation, lack of sister chromatid resolution, and lack of chromosome individualization.

Chromosome cohesion - rings, knots, orcs and fellowship.

Sister-chromatid cohesion is essential for accurate chromosome segregation. A key discovery towards our understanding of sister-chromatid cohesion was made 10 years ago with the identification of cohesins. Since then, cohesins have been shown to be involved in cohesion in numerous organisms, from yeast to mammals.

Cohesin is dispensable for centromere cohesion in human cells.

Background: Proper regulation of the cohesion at the centromeres of human chromosomes is essential for accurate genome transmission. Exactly how cohesion is maintained and is then dissolved in anaphase is not understood.

Principal Findings: We have investigated the role of the cohesin complex at centromeres in human cells both by depleting cohesin subunits using RNA interference and also by expressing a non-cleavable version of the Rad21 cohesin protein.

Regulation of centromeric cohesion by sororin independently of the APC/C.

Regulated separation of sister chromatids is the key event of mitosis. Sister chromatids remain cohered from the moment of DNA duplication until anaphase. Two known factors account for cohesion: DNA catenations and cohesin complexes.

PIASgamma is required for faithful chromosome segregation in human cells.

Background: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood.

Topoisomerase II checkpoints: universal mechanisms that regulate mitosis.

Checkpoint controls confer order to the cell cycle and help prevent genome instability. Here we discuss the Topoisomerase II (Decatenation) Checkpoint which functions to regulate mitotic progression so that chromosomes can be efficiently condensed in prophase and can be segregated with high fidelity in anaphase.

A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin.

Topoisomerase II (Topo II) performs topological modifications on double-stranded DNA molecules that are essential for chromosome condensation, resolution, and segregation. In mammals, G2 and metaphase cell cycle delays induced by Topo II poisons have been proposed to be the result of checkpoint activation in response to the catenation state of DNA. However, the apparent lack of such controls in model organisms has excluded genetic proof that Topo II checkpoints exist and are separable from the conventional DNA damage checkpoint controls.

Evidence that the yeast spindle assembly checkpoint has a target other than the anaphase promoting complex.

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null.

Anaphase promoting complex or cyclosome?

The anaphase promoting complex/cyclosome (APC/C) was initially described as a multi-subunit protein complex that ubiquitinates anaphase inhibitors thus targeting them for destruction by proteasomes to initiate loss of sister chromatid cohesion. However, recent studies have identified important new functions of the APC/C. Moreover, sister centromere separation can occur in the absence of APC/C activity in mammals, indicating that anaphase onset might be triggered by multiple factors.

Proteasome activity is required for centromere separation independently of securin degradation in human cells.

Loss of centromere cohesion during anaphase in human cells is regulated by the spindle assembly checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). APC-Cdc20 adds ubiquitin chains to securin inducing its destruction by the proteasome and these events correlate with the loss of sister chromatid cohesion and the onset of anaphase. But whether securin destruction is necessary and sufficient for anaphase initiation is not clear.

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC).

Separase is required at multiple pre-anaphase cell cycle stages in human cells.

The yeast separase proteins Esp1 and Cut1 are required for loss of sister chromatid cohesion that occurs at the moment of anaphase onset. Circumstantial evidence has linked human separase to centromere separation at anaphase, but a direct test that the role of this enzyme is functionally conserved with the yeast proteins is lacking. Here we describe the effects of separase depletion from human cells using RNA interference.

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis.

Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5 mM caffeine.

Roles of polo-like kinase 1 in the assembly of functional mitotic spindles.

Background: The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation.

Regulation of sister chromatid cohesion between chromosome arms.

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion.