Proliferative Cell Fate Research Articles

Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events

Understanding the dynamics of intracellular signaling pathways, such as ERK1/2 (ERK) and Akt1/2 (Akt), in the context of cell fate decisions is important for advancing our knowledge of cellular processes and diseases, particularly cancer. While previous studies have established associations between ERK and Akt activities and proliferative cell fate, the heterogeneity of single-cell responses adds complexity to this understanding. This study employed a data-driven approach to address this challenge, developing machine learning models trained on a dataset of growth factor-induced ERK and Akt activity time courses in single cells, to predict cell division events. The most predictive models were developed by applying discrete wavelet transforms (DWTs) to extract low-frequency features from the time courses, followed by using Ensemble Integration, a data integration and predictive modeling framework. The results demonstrated that these models effectively predicted cell division events in MCF10A cells (F-measure=0.524, AUC=0.726). ERK dynamics were found to be more predictive than Akt, but the combination of both measurements further enhanced predictive performance. The ERK model`s performance also generalized to predicting division events in RPE cells, indicating the potential applicability of these models and our data-driven methodology for predicting cell division across different biological contexts. Interpretation of these models suggested that ERK dynamics throughout the cell cycle, rather than immediately after growth factor stimulation, were associated with the likelihood of cell division. Overall, this work contributes insights into the predictive power of intra-cellular signaling dynamics for cell fate decisions, and highlights the potential of machine learning approaches in unraveling complex cellular behaviors.

Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP.

Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with low Delta/Notch signaling (differentiative cell fate). The cell polarity protein partitioning-defective 3 (Par-3) is critical for regulating this process. To understand how polarized Par-3 on the cell cortex can lead to differential Notch activity in the nuclei of daughter cells, we combined an anti-Delta D (Dld) -atto 647N antibody uptake assay with label retention expansion microscopy (LR-ExM), to obtain high resolution immunofluorescent images of Par-3, dynein light intermediate chain 1 (Dlic1), and Dld endosomes in mitotic RGPs. We then developed a protocol for analyzing the colocalization of Par-3, Dlic1, and endosomal DeltaD, using JACoP (Just Another Co-localization Plugin) in ImageJ software (Bolte and Cordelières, 2006). Through such analyses, we have shown that cytosolic Par-3 is associated with Dlic1 on Dld endosomes. Our work demonstrates a direct involvement of Par-3 in dynein-mediated polarized transport of Notch signaling endosomes. This bio-protocol may be generalizable for analysis of protein co-localization in any cryosectioned and immunostained tissue samples.

Subunits of the DNA polymerase alpha‐primase complex promote Notch‐mediated proliferation with discrete and shared functions in C. elegans germline

Notch receptor signaling is a highly conserved cell communication system in most multicellular organisms and plays a critical role at several junctures in animal development. In Caenorhabditis elegans,GLP-1/Notch signaling is essential for both germline stem cell maintenance and germ cell proliferation during gonad development. Here, we show that subunits (POLA-1, DIV-1, PRI-1, and PRI-2) of the DNA polymerase alpha-primase complex are required for germ cell proliferation in response to GLP-1/Notch signaling in different tissues at different developmental stages. Specifically, genetic and functional analyses demonstrated that (a) maternally contributed DIV-1 (regulatory subunit) is indispensable non-cell autonomously for GLP-1/Notch-mediated germ cell proliferation during early larval development, whereas POLA-1 (catalytic subunit) and two primase subunits, PRI-1 and PRI-2, do not appear to be essential; (b) germline POLA-1, PRI-1, and PRI-2 play a crucial role in GLP-1/Notch-mediated maintenance of proliferative cell fate during adulthood, while DIV-1 is dispensable; and (c) germline POLA-1, DIV-1, PRI-1, and PRI-2 function in tandem with PUF (Pumilio/FBF) RNA-binding proteins to maintain germline stem cells in the adult gonad. These findings suggest that the subunits of the DNA polymerase alpha-primase complex exhibit both discrete and shared functions in GLP-1/Notch or PUF-mediated germ cell dynamics in C. elegans. These findings link the biological functions of DNA replication machineries to signals that maintain a stem cell population, and may have further implications for Notch-dependent tumors.

Small GTPase RhoE/Rnd3 Is a Critical Regulator of Notch1 Signaling

Aberrations of Notch signaling have been implicated in a variety of human cancers. Oncogenic mutations in NOTCH1 are common in human T-cell leukemia and lymphomas. However, loss-of-function somatic mutations in NOTCH1 arising in solid tumors imply a tumor suppressor function, which highlights the need to understand Notch signaling more completely. Here, we describe the small GTPase RhoE/Rnd3 as a downstream mediator of Notch signaling in squamous cell carcinomas (SCC) that arise in skin epithelia. RhoE is a transcriptional target of activated Notch1, which is attenuated broadly in SCC cells. RhoE depletion suppresses Notch1-mediated signaling in vitro, rendering primary keratinocytes resistant to Notch1-mediated differentiation and thereby favoring a proliferative cell fate. Mechanistic investigations indicated that RhoE controls a key step in Notch1 signaling by mediating nuclear translocation of the activated portion of Notch1 (N1IC) through interaction with importins. Our results define RhoE as a Notch1 target that is essential for recruitment of N1IC to the promoters of Notch1 target genes, establishing a regulatory feedback loop in Notch1 signaling. This molecular circuitry may inform distinct cell fate decisions to Notch1 in epithelial tissues, where carcinomas such as SCC arise.

Temporal Patterns of Cortical Proliferation of Glial Cell Populations after Traumatic Brain Injury in Mice

TBI (traumatic brain injury) triggers an inflammatory cascade, gliosis and cell proliferation following cell death in the pericontusional area and surrounding the site of injury. In order to better understand the proliferative response following CCI (controlled cortical impact) injury, we systematically analyzed the phenotype of dividing cells at several time points post-lesion. C57BL/6 mice were subjected to mild to moderate CCI over the left sensory motor cortex. At different time points following injury, mice were injected with BrdU (bromodeoxyuridine) four times at 3-h intervals and then killed. The greatest number of proliferating cells in the pericontusional region was detected at 3 dpi (days post-injury). At 1 dpi, NG2+ cells were the most proliferative population, and at 3 and 7 dpi the Iba-1+ microglial cells were proliferating more. A smaller, but significant number of GFAP+ (glial fibrillary acidic protein) astrocytes proliferated at all three time points. Interestingly, at 3 dpi we found a small number of proliferating neuroblasts [DCX+ (doublecortin)] in the injured cortex. To determine the cell fate of proliferative cells, mice were injected four times with BrdU at 3 dpi and killed at 28 dpi. Approximately 70% of proliferative cells observed at 28 dpi were GFAP+ astrocytes. In conclusion, our data suggest that the specific glial cell types respond differentially to injury, suggesting that each cell type responds to a specific pattern of growth factor stimulation at each time point after injury.

Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline

The C. elegans germline provides an excellent model for analyzing the regulation of stem cell activity and the decision to differentiate and undergo meiotic development. The distal end of the adult hermaphrodite germline contains the proliferative zone, which includes a population of mitotically cycling cells and cells in meiotic S phase, followed by entry into meiotic prophase. The proliferative fate is specified by somatic distal tip cell (DTC) niche-germline GLP-1 Notch signaling through repression of the redundant GLD-1 and GLD-2 pathways that promote entry into meiosis. Here, we describe characteristics of the proliferative zone, including cell cycle kinetics and population dynamics, as well as the role of specific cell cycle factors in both cell cycle progression and the decision between the proliferative and meiotic cell fate. Mitotic cell cycle progression occurs rapidly, continuously, with little or no time spent in G1, and with cyclin E (CYE-1) levels and activity high throughout the cell cycle. In addition to driving mitotic cell cycle progression, CYE-1 and CDK-2 also play an important role in proliferative fate specification. Genetic analysis indicates that CYE-1/CDK-2 promotes the proliferative fate downstream or in parallel to the GLD-1 and GLD-2 pathways, and is important under conditions of reduced GLP-1 signaling, possibly corresponding to mitotically cycling proliferative zone cells that are displaced from the DTC niche. Furthermore, we find that GLP-1 signaling regulates a third pathway, in addition to the GLD-1 and GLD-2 pathways and also independent of CYE-1/CDK-2, to promote the proliferative fate/inhibit meiotic entry.

A role for dynein in the inhibition of germ cell proliferative fate.

During normal development as well as in diseased states such as cancer, extracellular "niches" often provide cues to proximal cells and activate intracellular pathways. Activation of such signaling pathways in turn instructs cellular proliferation and differentiation. In the Caenorhabditis elegans gonad, GLP-1/Notch signaling instructs germ line stem cells to self-renew through mitotic cell division. As germ cells progressively move out of the niche, they differentiate by entering meiosis and eventually form gametes. In this model system, we uncovered an unexpected role for the dynein motor complex in promoting normal differentiation of proliferating germ cells. We demonstrate that dynein light chain 1 (DLC-1) and its partner, dynein heavy chain 1, inhibit the proliferative cell fate, in part through regulation of METT-10, a conserved putative methyltransferase. We show that DLC-1 physically interacts with METT-10 and promotes both its overall levels and nuclear accumulation. Our results add a new dimension to the processes controlled by the dynein motor complex, demonstrating that dynein can act as an antiproliferative factor.

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

In teleost fish, unlike other vertebrates, the retina continues to grow throughout the animal's life both by stretching of the mature tissue and by the addition of new cells. Following larval development, new retinal cell birth is known to occur in a rim at the periphery of the mature retina and in the outer nuclear layer (ONL). We have now found that cell birth and proliferation also occurs in the inner nuclear layer (INL) of the mature fish retina. In rainbow trout (Onchoryncus mykiss), proliferative cells exist in the INL of fish of all ages, at least up to 2 years posthatching. The proliferative cells form clusters in the INL that align in radial columns, reaching from the inner to the outer plexiform layers. The density of proliferative cell clusters changes along the equatorial plane of the retina and is highest near both the nasal and temporal poles. Our data suggest that, after birth, the proliferative cells migrate away from the INL and into the ONL, with a half-time of about 3 days, and their cell bodies can be seen in the outer plexiform layer. Once they are in the ONL, the proliferative cells continue to divide and likely give rise to the precursor cells that differentiate into new rod photoreceptors.

Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey

The nature of proliferative cells in the subgranular zone (SGZ) of the hippocampal region and the fate of their progeny was analyzed by 3H-thymidine (3H-TdR) autoradiography combined with immunocytochemistry at the light and electron microscopic levels in 18 rhesus monkeys ranging in age from late gestation to 17 years. Our analysis indicates that, during the last quarter of gestation and the first 3 postnatal months, the SGZ produces both glial and neuronal cells. These 2 major classes of cells originate from the 2 precursor lines and, following their mitotic division, migrate to the granular layer. During the juvenile period (4-6 months of age), neuronal production tapers off and most postmitotic cells remaining within the SGZ differentiate into glial elements. In postpubertal animals (3 years and older), the 3H-TdR-labeled cells in the dentate gyrus belong to several non-neuronal classes. The largest group was immunoreactive to the glial fibrillary acidic protein (GFAP) at both the light and electron microscopic levels, indicating their astrocytic nature. The remaining 3H-TdR-labeled, GFAP-negative cells had ultra-structural characteristics of either microglia, oligodendroglia, or their progenitory stem cells. Therefore, there is a continuing addition and/or turnover of the glial cells in the dentate gyrus of sexually mature monkeys, but, in contrast to the massive neurogenesis reported in adult rodents, the production of new neurons could not be detected after puberty. The significance of a stable population of neurons in the hippocampal formation of mature primates is discussed in relation to its possible function in memory.