Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Universal sample preparation method for proteome analysis

https://cyberleninka.org/article/n/1346988.pdf

Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

It has become clear that the role of centrosomes extends well beyond that of important microtubule organizers. There is increasing evidence that they also function as coordination centres in eukaryotic cells, at which specific cytoplasmic proteins interact at high concentrations and important cell decisions are made. Accordingly, hundreds of proteins are concentrated at centrosomes, including cell cycle regulators, checkpoint proteins and signalling molecules. Nevertheless, several observations have raised the question of whether centrosomes are essential for many cell processes. Recent findings have shed light on the functions of centrosomes in animal cells and on the molecular mechanisms of centrosome assembly, in particular during mitosis. These advances should ultimately allow the in vitro reconstitution of functional centrosomes from their component proteins to unlock the secrets of these enigmatic organelles.

Centrosome function and assembly in animal cells

Centrioles are conserved microtubule-based organelles that form the core of the centrosome and act as templates for the formation of cilia and flagella. Centrioles have important roles in most microtubule-related processes, including motility, cell division and cell signalling. To coordinate these diverse cellular processes, centriole number must be tightly controlled. In cycling cells, one new centriole is formed next to each pre-existing centriole in every cell cycle. Advances in imaging, proteomics, structural biology and genome editing have revealed new insights into centriole biogenesis, how centriole numbers are controlled and how alterations in these processes contribute to diseases such as cancer and neurodevelopmental disorders. Moreover, recent work has uncovered the existence of surveillance pathways that limit the proliferation of cells with numerical centriole aberrations. Owing to this progress, we now have a better understanding of the molecular mechanisms governing centriole biogenesis, opening up new possibilities for targeting these pathways in the context of human disease.

Once and only once: mechanisms of centriole duplication and their deregulation in disease

Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle–regulated accumulation of STIL.

/pdf/binding-of-stil-to-plk4-activates-kinase-activity-to-promote-38fo0io245.pdf

Binding of STIL to Plk4 activates kinase activity to promote centriole assembly.

Precise regulation of centrosome number is critical for accurate chromosome segregation and the maintenance of genomic integrity. In nontransformed cells, centrosome loss triggers a p53-dependent surveillance pathway that protects against genome instability by blocking cell growth. However, the mechanism by which p53 is activated in response to centrosome loss remains unknown. Here, we have used genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 after centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth after a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells that have an increased propensity for mitotic errors.

/pdf/a-usp28-53bp1-p53-p21-signaling-axis-arrests-growth-after-30qnx5w2an.pdf

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis.

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure.

/pdf/p53-protects-against-genome-instability-following-centriole-4t40s2soqr.pdf

p53 protects against genome instability following centriole duplication failure

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain–containing gene, specifically Kelch domain containing 8A (KLHDC8A) with a previously unknown function as an epigenetically driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound chaperonin-containing TCP1 (CCT) to promote the assembly of primary cilia to activate hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and hedgehog signaling. Combinatorial targeting of Aurora B/C kinase and hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as what we believe to be a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.

http://www.jci.org/articles/view/163592/files/pdf

Superenhancer activation of KLHDC8A drives glioma ciliation and hedgehog signaling


 
 
 The Hedgehog pathway directs gene expression programs that are essential for glioblastoma stem cells in vitro, but a clinical trial suggested Hedgehog pathway inhibition does not improve glioblastoma outcomes (ABTC-0904). In pancreatic and bladder cancer, Hedgehog signaling through the tumor microenvironment induces stromal morphogens to inhibit cancer stem cell proliferation. Thus, we hypothesized Hedgehog signaling through the glioblastoma microenvironment may inhibit glioblastoma stem cell proliferation in vivo.
 
 
 
 Hedgehog signaling was studied using patient-derived, fluorescently-labeled IDH wild-type glioblastoma cells (GBM6, SF11956, SF11907, GPMP004, GPMP005) in vitro, in vivo after intracranial implantation in mice, or in 3D co-culture with iPSC-derived organoids comprised of human astrocytes or neurons. Genetic loss-of-function experiments were performed using CRISPR interference. Cell proliferation, stem cell marker expression, Sonic Hedgehog (SHH) expression, or primary cilia architecture were assessed using immunofluorescence, confocal microscopy, or live-cell imaging. Hedgehog signaling or morphogen expression were assessed using QPCR or single-cell RNA sequencing. Pharmacologic experiments were performed using vismodegib to inhibit the Hedgehog pathway, or FK506 to induce stromal morphogen expression.
 
 
 
 Vismodegib increased glioblastoma cell proliferation and stem cell marker expression in vivo or in co-culture with astrocyte organoids. Single-cell RNA sequencing demonstrated glioblastoma cell co-culture with astrocyte organoids induced Hedgehog target genes and stem cell markers in glioblastoma cells, or stromal morphogens in astrocytes, which also expressed primary cilia. CRISPRi suppression of SHH in glioblastoma cells increased glioblastoma cell proliferation and stem cell marker expression in co-culture with astrocyte organoids. Neither vismodegib nor SHH suppression induced glioblastoma cell proliferation or stem cell marker expression in vitro or in co-culture with neuron organoids. FK506 induced astrocyte morphogen expression, inhibiting glioblastoma cell proliferation, stem cell marker expression, and the effects of vismodegib in co-culture with astrocyte organoids.
 
 
 
 SHH from glioblastoma cells signals through astrocytes to inhibit cancer stem cell proliferation.


/pdf/tmic-43-hedgehog-ligands-from-glioblastoma-cells-induce-31eg4gug.pdf

Vikas Daggubati

Papers

Binding of STIL to Plk4 activates kinase activity to promote centriole assembly.

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis.

p53 protects against genome instability following centriole duplication failure

Superenhancer activation of KLHDC8A drives glioma ciliation and hedgehog signaling

Tmic-43. hedgehog ligands from glioblastoma cells induce astrocytes inhibition of cancer stem cells