Sander van den Heuvel (2005) WormBook "Cell-cycle regulation."

Sander Van Den Heuvel

[WormBook, 2005]

Cell-division control affects many aspects of development. Caenorhabditis elegans cell-cycle genes have been identified over the past decade, including at least two distinct Cyclin-Dependent Kinases (CDKs), their cyclin partners, positive and negative regulators, and downstream targets. The balance between CDK activation and inactivation determines whether cells proceed through G 1 into S phase, and from G 2 to M, through regulatory mechanisms that are conserved in more complex eukaryotes. The challenge is to expand our understanding of the basic cell cycle into a comprehensive regulatory network that incorporates environmental factors and coordinates cell division with growth, differentiation and tissue formation during development. Results from several studies indicate a critical role for CKI-1 , a CDK inhibitor of the Cip/Kip family, in the temporal control of cell division, potentially acting downstream of heterochronic genes and dauer regulatory pathways.

Sendinc E et al. (2015) Cell "Remodeling Your Way out of Cell Cycle."

Sendinc E, Shi Y, Jambhekar A

[Cell, 2015]

Throughout development, proliferative progenitors lose their mitotic potential, exit the cell cycle, and differentiate. In this issue, Ruijtenberg and van den Heuvel identify an important lineage-specific role for a SWI/SNF chromatin-remodeling complex that collaborates with core cell-cycle regulators to promote cell-cycle exit and terminal muscle cell differentiation.

Ruijtenberg, Suzan et al. (2013) International Worm Meeting "CRE-LoxP mediated gene inactivation to study the coordination between proliferation and differentiation."

Van den Heuvel, Sander, Ruijtenberg, Suzan

[International Worm Meeting, 2013]

The proper balance between proliferation and differentiation is critical for the formation and function of somatic cells, tissues and organs. During development, terminal differentiation usually coincides with permanent cell-cycle exit, while sustained proliferation is a hallmark of cancer cells. Despite their importance, the mechanisms that accomplish cell-cycle arrest in a developmental context are poorly understood. Proliferation and differentiation are tightly coordinated in C. elegans, as illustrated by its reproducible cell lineage and lack of mutants with severe over-proliferation phenotypes. Inactivation of lin-35 Rb leads to substantial hyperplasia only when combined with loss of other negative regulators of the cell cycle, such as fzr-1 Cdh1 or cki-1/2 Cyclin-dependent Kinase Inhibitors. Importantly, even lin-35;cki-1 and lin-35;fzr-1 double mutants form apparently normal post-mitotic differentiated cells. While this points to redundant control of cell-cycle arrest, essential functions in embryogenesis and maternal contribution complicate the genetic analysis of combined mutations. To be able to study cell-cycle arrest in post-embryonic cell lineages, we developed a CRE-LoxP based recombination system. This system combines two features: tissue-specific expression of the CRE recombinase induces lineage-specific inactivation of a gene of interest and, at the same time, causes a switch in fluorescent protein expression. This latter aspect visualizes recombination events and helps lineage tracing. After optimization and dependent on the promoter used, CRE induced recombination in many different tissues, with nearly 100% efficiency in the intestine and mesoblast lineages. Combination of lin-35 knock down and intestine-specific inactivation of fzr-1 Cdh1 by CRE-LoxP recombination resulted in overproliferation in the intestine to a similar extent as the lin-35;fzr-1 double mutant. Importantly, other tissues were unaffected and the knock-out animals were viable and fertile, in contrast to the double mutant. We currently focus on the mesoblast lineage to obtain a comprehensive understanding of the regulatory mechanisms that coordinate cell-cycle exit with muscle differentiation.

Wildwater, Marjolein et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "ALG-1/ Argonaut ensures proper development by coordinated regulation of cell polarity, cell-cell contacts, spindle positioning, and developmental timing"

Van den Heuvel, Sander, Sander, Nicholas, Wildwater, Marjolein

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Proper development of an organism requires both cell division and cell differentiation. Asymmetric cell division leads to the formation of two daughters with unequal fates. C. elegans epidermal seam cells are an excellent model system to study asymmetric division as seam cells divide in an asymmetrical manner once during every larval stage. To understand the regulatory mechanisms of asymmetric seam cell divisions we performed a mutagenesis screen to identify new components involved in asymmetric seam cell division control. We identified a mutation in alg-1 /Argonaut, an essential component of the RISC complex and important in regulation of miRNA function. With a newly developed in vivo time-lapse system for larvae we inve stigated the importance of alg-1 /miRNAs in asymmetric seam cell division control. We observed a sequential range of developmental aberrations, emphasizing the importance of miRNA function during C. elegans development. Reduction of alg-1 first causes mislocalisation of Wnt signaling pathway components and cell fate defects. Then, cell-cell contacts were lost, cell membranes showed hyper motility, cell shape of seam cells became increasingly round and cells started to divide in random directions. As a late effect, we observed developmental timing defects. We discovered that miRNAs responsible for regulation of developmental timing do not regulate division polarity or membrane integrity. We furthermore found epidermal seam cell shape to be critical in division orientation control by miRNAs. Our results highlight the important role of miRNAs during several steps of developmental cell division events and shows that like in mammalian systems miRNAs are crucial factors to re gulate development at different branch points.

Blackwell, Alexander et al. (2021) International Worm Meeting "Studying chromatin regulation at single cell resolution during C. elegans postembryonic development"

Gaarenstroom, Tessa, Van den Heuvel, Sander, Blackwell, Alexander

[International Worm Meeting, 2021]

Despite containing identical genomes, developing cells differentiate into a plethora of diverse cell types. Distinct patterns of gene expression now form the basis for classifying different cell fates. How the combinatorial activity of transcription factors, chromatin regulators and histone modifications achieve the proper spatiotemporal patterns of gene expression is a major question in developmental biology. Biologists increasingly appreciate the need to investigate gene expression regulation at the single-cell level because much heterogeneity and complexity is lost when averaging across populations of cells. However, profiling chromatin at the single cell level is challenging due to limited input material. Chromatin immunocleavage with sequencing (ChIC-seq) is an efficient method to study chromatin modifications from low input samples. ChIC-seq utilises antibody targeted micrococcal nucleases, leading to controlled, binding-dependent enzymatic digestion of DNA. This releases short fragments which become preferentially incorporated during library preparation and enables high resolution mapping of genomic positions. Crucially, the absence of crosslinking and immunoprecipitation steps, required in less sensitive techniques such as ChIP-seq, leads to minimal material loss. Recently, ChIC-seq was used to profile histone modifications in single human cells [1]. Adapting ChIC-seq to profile histone modifications in C. elegans will provide a powerful tool for studying the epigenetic regulation of development. Here, we present progress in optimising ChIC-seq for profiling chromatin modifications at single-cell level across a developmental time-course in C. elegans. Specifically, we combine Cre/Lox lineage tracing with cell isolation and FACS procedures in order to isolate postembryonic mesoderm cells. Following prolonged quiescence, the mesoblast precursor resumes proliferation and produces fourteen muscle cells, two scavenger cells, and two migratory bipotent myoblasts over 24-hours. By profiling chromatin modifications at high temporal resolution, we aim to reveal regulatory processes controlling cellular proliferation and differentiation. This work will shed light on how epigenetic modifications contribute to cellular decision making in a living animal. [1] Ku WL, Nakamura K, Gao W, Cui K, Hu G, Tang Q, Ni B, Zhao K. (2019) Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification. Nature Methods, vol. 16, pages 323-325.

Portegijs, Vincent et al. (2021) International Worm Meeting "Multiple levels of regulation ensure robust cell cycle exit during C. elegans vulva formation"

Godfrey, Molly, Portegijs, Vincent, Van den Heuvel, Sander

[International Worm Meeting, 2021]

Regulated cell cycle withdrawal is crucial for cell differentiation and tissue homeostasis, with impaired cell cycle exit leading to hyperplasia, a hallmark of cancer. The reproducible pattern of cell proliferation and differentiation in C. elegans provides an excellent model to study the regulatory pathways that control cell cycle arrest. Using tissue-specific gene knockout combined with lineage tracing, we explored whether known cell cycle inhibitors and tumor suppressor genes regulate cell cycle exit of the epidermal vulva precursor cells (VPCs). Lineage-specific knockout of cki-1, the main C. elegans CIP/KIP cell-cycle inhibitor, resulted in a surprisingly weak extra vulval cell division phenotype. We found that a second CIP/KIP inhibitor, cki-2, becomes transcriptionally upregulated in these cki-1 knockout animals. Simultaneous knockout of both CKIs substantially increased the number of extra vulva cells. In addition to cki-2, we discovered that cep-1 p53 and daf-3 Smad4 contribute to proper cell cycle arrest of cki-1 knockout vulval cells. Genetic analyses support a model in which these transcriptional regulators act upstream of cki-2 to promote cki-2 expression, which is critical only when cki-1 is absent. Furthermore, a forward genetic screen identified the HECT-domain ubiquitin ligase UBR-5 as another factor contributing to the timely VPC cell division arrest. The ubr-5 overproliferation phenotype was markedly enhanced when combined with cki-1 knockout, indicating that UBR-5 may act in parallel to CKI-1. Notably, all tested combinations of negative cell-cycle regulator knockout/knockdown resulted in limited increases in vulval cell numbers. Our data support that multiple redundant levels of regulation ensure remarkably robust control of cell cycle withdrawal of C. elegans vulval cells.

Galli, Matilde et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A quantitative mass spec approach reveals phosphorylation of the spindle positioning protein LIN-5 by the anterior PAR complex"

Heck, Albert, Galli, Matilde, Munoz, Javier, Van den Heuvel, Sander

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During asymmetric cell division, a cell first acquires an axis of polarity, and then aligns the mitotic spindle along this polarity axis to generate daughter cells with different fates. Although it is becoming clear how polarity cues are established, it is unknown how these cues regulate the position of the mitotic spindle to direct the plane of division. The conserved LIN-5/GPR/G? complex is thought to act downstream of polarity signals to generate microtubule pulling forces, but a direct link between polarity proteins and the LIN-5/GPR/G? complex has not been identified. To determine whether this complex is directly controlled by polarity proteins, either in protein composition or by post-translational modifications, we set up a quantitative mass spectrometry approach. For this, early C. elegans embryos were differentially labeled with either 15N or 14N isotopes and treated with either control RNAi or RNAi against the polarity protein kinase pkc-3. LIN-5 immunoprecipitations from the 14N/15N mixed lysates were then analyzed by mass spectrometry. Strikingly, depletion of pkc-3 resulted in a six-fold reduction in phosphorylation of four residues on LIN-5, while all other phosphorylation sites were unaltered. Thus, PKC-3 promotes the phosphorylation of LIN-5 in early embryonic divisions. Using in vitro kinase assays with recombinant LIN-5 we found that PKC-3 directly phosphorylates these residues on LIN-5. To determine the role of these phosphorylations during spindle positioning, we are currently analyzing non-phosphorylatable and phospho-mimicking LIN-5 mutants. Preliminary results indicate that LIN-5 phospho-mimicking mutants have reduced spindle pulling forces and have defects in asymmetric cell division. Together, these results suggest that PKC-3 controls spindle positioning through direct phosphorylation of LIN-5.

Harterink, Martin et al. (2013) International Worm Meeting "Neuronal microtubule organization in C. elegans."

Hoogenraad, Casper, Van den Heuvel, Sander, de Haan, Bart, Harterink, Martin

[International Worm Meeting, 2013]

In polarized cells, such as neurons, the microtubule cytoskeleton offers tracks to transport various cargos along axonal and dendritic projections. Recent data indicate that differences in microtubule organization within axons and dendrites enable molecular motors to sort cargo to specific directions and establish and maintain neuronal polarity. However how these differences are established is hardly understood. C. elegans has relatively simple neurons which offer a great starting point to understand the basics of this microtubule organization. Using GFP tagged end-binding proteins to visualize microtubule growth we have compared several classes of neurons with different characteristics (such as ciliated vs unciliated and branched vs unbrached). Here we will present a preliminary analyses of the neuronal microtubule organization of wildtype as well as in several mutants.

Portegijs, Vincent et al. (2015) International Worm Meeting "Regulation of spindle positioning through phosphorylation of LIN-5 in asymmetric cell division."

Fielmich, Lars-Eric, Van den Heuvel, Sander, Portegijs, Vincent, Galli, Matilde

[International Worm Meeting, 2015]

Asymmetric cell division is required throughout development to generate cells of unequal size, composition and fate. The correct distribution of cell fate determinants depends on the plane of cell cleavage, which is determined by the position of the mitotic spindle. Spindle positioning is mediated by a conserved Galpha-GPR-1/2­-LIN-5 complex at the cortex, which interacts through dynein with astral microtubules and thereby promotes the generation of pulling forces. We aim to understand how the developmental regulation of cortical pulling forces determines the plane of cell cleavage and contributes to asymmetric cell division.Previous studies of our lab identified four amino acids in the C-terminal domain of LIN-5 that are phosphorylated by the polarity kinase PKC-3. This phosphorylation leads to reduced pulling forces in the anterior, but additional mechanisms contribute to asymmetric spindle positioning. In recent studies, we identified additional phosphorylations of LIN-5 with critical functions in vivo. Moreover, we defined the LIN-5 C-terminal domain that mediates interaction with GPR-1. Hereto, we used reverse yeast two-hybrid screening to identify single amino acid changes in LIN-5 that prevent GPR-1 binding. All of the identified mutations are confined to a 30 amino-acid domain located upstream of the PKC-3 phosphorylated residues of LIN-5. This GPR-1 binding region contains two additional in vivo phosphorylated LIN-5 residues. We used Cas9/CRISPR-mediated genome editing to substitute these amino acids for non-phosphorylatable alanine or phosphomimetic glutamic acid / aspartic acid. Combined with UV-laser spindle severing experiments, we found that phosphorylation of these residues contributes to GPR-1 binding and cortical pulling forces. In a parallel study, we identified residues in the N-terminus of LIN-5 that are phosphorylated by CDK-1. Again, examination of Cas9/CRISPR-mediated knock-in alleles revealed that these N-terminal residues are critical for LIN-5 function. Taken together, our data show that phosphorylation of LIN-5 contributes to the spatiotemporal regulation of the cortical pulling forces that position the mitotic spindle.

Fielmich, Lars-Eric et al. (2017) International Worm Meeting "Spatiotemporal control of gene knock-out and protein-protein heterodimerization to study mitotic spindle positioning."

Van den Heuvel, Sander, Schmidt, Ruben, Fielmich, Lars-Eric, Akhmanova, Anna

[International Worm Meeting, 2017]

Tissue development and homeostasis depend on the correct axis and plane of cell division, which are determined by the position of the mitotic spindle. Studies of the C. elegans one-cell embryo have provided much insight in the regulation of spindle positioning during asymmetric cell division. Pulling forces that act from the cell cortex on astral microtubules were found to position the spindle. These pulling forces depend on a force-generating complex (FG) that anchors the dynein motor to the cortex. The FG is a conserved complex consisting of a membrane-bound Ga protein that, in the GDP-bound form, binds the GPR-1/2 linker protein, which interacts with the LIN-5 coiled-coil protein. The discovery of a non-canonical role of Ga as a membrane-anchor for the FG was surprising at the time, and still is only partly understood. The Guanine-nucleotide Exchange Factor (GEF) RIC-8 and GTPase-Activating Protein (GAP) RGS-7 regulate the GDP/GTP cycle of Ga and are also required for proper force generation. It remains unclear how this corresponds to Ga functioning as a membrane anchor. Hampering analysis, complete inactivation of ric-8 and rgs-7 appears difficult to achieve by RNAi and null mutants are not viable. We therefore developed an inducible, tissue-specific gene knock-out system based on Cre-Lox recombination. We introduced Lox sites in the endogenous ric-8 and rgs-7 loci to enable KO of the genes. To assure spatiotemporal control of the KO, we use a two-component recombinase system. Thus, gene KO can be induced in the tissue of choice, for example the germline, while maintaining a healthy strain. A complementary approach focuses on the individual functions of FG components. Hereto, we use the light-inducible heterodimerization of LOV and ePDZ to reconstitute cortical FG complexes that lack one or several components. Using a PH::LOV as membrane anchor and an ePDZ::dynein knock-in, we were able to recruit endogenous dynein directly to the membrane and bypass the FG. This was insufficient for pulling force generation and suggests that the FG is more than a dynein anchor. To test the role of Ga signaling, we generated and are currently analyzing an ePDZ::GPR-1 knock-in to reconstitute a cortical FG that lacks the Ga component. We will present our latest results at the meeting.