Spatial mechanisms of quality control during chaperone-mediated assembly of the proteasome.

Cellular protein degradation requires a complex molecular machine, the proteasome. To mitigate the fundamental challenge of assembling the 66-subunit proteasome, cells utilize dedicated chaperones to order subunit addition. However, recent evidence suggests that proteasome assembly is not simply a series of subunit additions, but each step may be scrutinized so that only correct assembly events advance to proteasomes.

Assembly checkpoint of the proteasome regulatory particle is activated by coordinated actions of proteasomal ATPase chaperones.

The proteasome holoenzyme regulates the cellular proteome via degrading most proteins. In its 19-subunit regulatory particle (RP), a heterohexameric ATPase enables protein degradation by injecting protein substrates into the core peptidase. RP assembly utilizes "checkpoints," where multiple dedicated chaperones bind to specific ATPase subunits and control the addition of other subunits.

Preclinical and Dose-Finding Phase I Trial Results of Combined Treatment with a TORC1/2 Inhibitor (TAK-228) and Aurora A Kinase Inhibitor (Alisertib) in Solid Tumors.

Purpose: The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors.

Experimental Design: TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD).

Two alternative mechanisms regulate the onset of chaperone-mediated assembly of the proteasomal ATPases.

The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as 'Rpt ring', assembles through six ATPase subunits (Rpt1-Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2, and Hsm3).

Loss of p53 expression in cancer cells alters cell cycle response after inhibition of exportin-1 but does not prevent cell death.

The tumor suppressor protein p53 is central to the cellular stress response and may be a predictive biomarker for cancer treatments. Upon stress, wildtype p53 accumulates in the nucleus where it enforces cellular responses, including cell cycle arrest and cell death. p53 is so dominant in its effects, that p53 enforcement - or - restoration therapy is being studied for anti-cancer therapy.

Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos.

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings.

Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor.

Longitudinal tracking is a powerful approach to understand the biology of single cells. In cancer therapy, outcome is determined at the molecular and cellular scale, yet relationships between cellular response and cell fate are often unknown. The selective inhibitor of nuclear export, selinexor, is in development for the treatment of various cancers.

In vivo cell-cycle profiling in xenograft tumors by quantitative intravital microscopy.

Quantification of cell-cycle state at a single-cell level is essential to understand fundamental three-dimensional (3D) biological processes such as tissue development and cancer. Analysis of 3D in vivo images, however, is very challenging. Today's best practice, manual annotation of select image events, generates arbitrarily sampled data distributions, which are unsuitable for reliable mechanistic inferences.

Single-cell pharmacokinetic imaging reveals a therapeutic strategy to overcome drug resistance to the microtubule inhibitor eribulin.

Eribulin mesylate was developed as a potent microtubule-targeting cytotoxic agent to treat taxane-resistant cancers, but recent clinical trials have shown that it eventually fails in many patient subpopulations for unclear reasons. To investigate its resistance mechanisms, we developed a fluorescent analog of eribulin with pharmacokinetic (PK) properties and cytotoxic activity across a human cell line panel that are sufficiently similar to the parent drug to study its cellular PK and tissue distribution. Using intravital imaging and automated tracking of cellular dynamics, we found that resistance to eribulin and the fluorescent analog depended directly on the multidrug resistance protein 1 (MDR1).

Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction.

Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1.

Rapid induction of apoptosis during Kinesin-5 inhibitor-induced mitotic arrest in HL60 cells.

Small molecule inhibitors of Kinesin-5 (K5Is) that arrest cells in mitosis with monopolar spindles are promising anti-cancer drug candidates. Clinical trials of K5Is revealed dose-limiting neutropenia, or loss of neutrophils, for which the molecular mechanism is unclear. We investigated the effects of a K5I on HL60 cells, a human promyelocytic leukemia cell line that is often used to model dividing neutrophil progenitors in cell culture.

Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics.

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy).

An intermittent live cell imaging screen for siRNA enhancers and suppressors of a kinesin-5 inhibitor.

Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers.

Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly.

Current antimitotics work by perturbing spindle assembly, which activates the spindle assembly checkpoint, causes mitotic arrest, and triggers apoptosis. Cancer cells can resist such killing by premature exit, before cells initiate apoptosis, due to a weak checkpoint or rapid slippage. We reasoned blocking mitotic exit downstream of the checkpoint might circumvent this resistance.

Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate.

Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis.

Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5.

To improve cancer chemotherapy, we need to understand the mechanisms that determine drug sensitivity in cancer and normal cells. Here, we investigate this question across a panel of 11 cell lines at a phenotypic and molecular level for three antimitotic drugs: paclitaxel, nocodazole, and an inhibitor of kinesin-5 (also known as KSP, Eg5, Kif11). Using automated microscopy with markers for mitosis and apoptosis (high content screening), we find that the mitotic arrest response shows relatively little variation between cell types, whereas the tendency to undergo apoptosis shows large variation.

Get off my back! Rapid receptor internalization through circular dorsal ruffles.

Internalization and subsequent trafficking of receptor tyrosine kinases (RTKs) play an important role in the modulation of growth factor-stimulated signaling events that affect different cellular processes, from cell growth and mitosis to motility and invasion. The intracellular transport of these receptors has traditionally been viewed as being initiated via clathrin-coated pits. However, nonclathrin pathways have been implicated as well, although these remain poorly understood.

A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization.

Cells form transient, circular dorsal ruffles or "waves" in response to stimulation of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor. These dynamic structures progress inward on the dorsal surface and disappear, occurring concomitantly with a marked reorganization of F-actin. The cellular function of these structures is largely unknown.

Actin and Arf1-dependent recruitment of a cortactin-dynamin complex to the Golgi regulates post-Golgi transport.

Cortactin is an actin-binding protein that has recently been implicated in endocytosis. It binds directly to dynamin-2 (Dyn2), a large GTPase that mediates the formation of vesicles from the plasma membrane and the Golgi. Here we show that cortactin associates with the Golgi to regulate the actin- and Dyn2-dependent transport of cargo.

Caveolin-1 interacts directly with dynamin-2.

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins.

Foot and mouth: podosomes, invadopodia and circular dorsal ruffles.

The plasma membrane of many motile cells undergoes highly regulated protrusions and invaginations that support the formation of podosomes, invadopodia and circular dorsal ruffles. Although they are similar in appearance and in their formation--which is mediated by a highly conserved actin-membrane apparatus--these transient surface membrane distortions are distinct. Their function is to help the cell as it migrates, attaches and invades.

Cdc42 and the actin-related protein/neural Wiskott-Aldrich syndrome protein network mediate cellular invasion by Cryptosporidium parvum.

Cryptosporidium parvum invasion of epithelial cells involves host cell membrane alterations which require a remodeling of the host cell actin cytoskeleton. In addition, an actin plaque, possibly associated with the dense-band region, forms within the host cytoplasm at the host-parasite interface. Here we show that Cdc42 and RhoA, but not Rac1, members of the Rho family of GTPases, are recruited to the host-parasite interface in an in vitro model of human biliary cryptosporidiosis.

A dynamin-cortactin-Arp2/3 complex mediates actin reorganization in growth factor-stimulated cells.

The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor.