Mitotic stress is an integral part of the oncogene-induced senescence program that promotes multinucleation and cell cycle arrest

Oncogene-induced senescence (OIS) is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells

Abrupt onset of mutations in a developmentally regulated gene during terminal differentiation of post-mitotic photoreceptor neurons in mice.

For sensitive detection of rare gene repair events in terminally differentiated photoreceptors, we generated a knockin mouse model by replacing one mouse rhodopsin allele with a form of the human rhodopsin gene that causes a severe, early-onset form of retinitis pigmentosa. The human gene contains a premature stop codon at position 344 (Q344X), cDNA encoding the enhanced green fluorescent protein (EGFP) at its 3' end, and a modified 5' untranslated region to reduce translation rate so that the mutant protein does not induce retinal degeneration. Mutations that eliminate the stop codon express a human rhodopsin-EGFP fusion protein (hRho-GFP), which can be readily detected by fluorescence microscopy. Spontaneous mutations were observed at a frequency of about one per retina; in every case, they gave rise to single fluorescent rod cells, indicating that each mutation occurred during or after the last mitotic division. Additionally, the number of fluorescent rods did not increase with age, suggesting that the rhodopsin gene in mature rod cells is less sensitive to mutation than it is in developing rods. Thus, there is a brief developmental window, coinciding with the transcriptional activation of the rhodopsin locus, in which somatic mutations of the rhodopsin gene abruptly begin to appear

Abrupt onset of mutations in a developmentally regulated gene during terminal differentiation of post-mitotic photoreceptor neurons in mice

For sensitive detection of rare gene repair events in terminally differentiated photoreceptors, we generated a knockin mouse model by replacing one mouse rhodopsin allele with a form of the human rhodopsin gene that causes a severe, early-onset form of retinitis pigmentosa. The human gene contains a premature stop codon at position 344 (Q344X), cDNA encoding the enhanced green fluorescent protein (EGFP) at its 3\u27 end, and a modified 5\u27 untranslated region to reduce translation rate so that the mutant protein does not induce retinal degeneration. Mutations that eliminate the stop codon express a human rhodopsin-EGFP fusion protein (hRho-GFP), which can be readily detected by fluorescence microscopy. Spontaneous mutations were observed at a frequency of about one per retina; in every case, they gave rise to single fluorescent rod cells, indicating that each mutation occurred during or after the last mitotic division. Additionally, the number of fluorescent rods did not increase with age, suggesting that the rhodopsin gene in mature rod cells is less sensitive to mutation than it is in developing rods. Thus, there is a brief developmental window, coinciding with the transcriptional activation of the rhodopsin locus, in which somatic mutations of the rhodopsin gene abruptly begin to appear

Spontaneous Changes in Taste Sensitivity of Single Units Recorded over Consecutive Days in the Brainstem of the Awake Rat

<div><p>A neuron’s sensitivity profile is fundamental to functional classification of cell types, and underlies theories of sensory coding. Here we show that gustatory neurons in the nucleus of the solitary tract (NTS) and parabrachial nucleus of the pons (PbN) of awake rats spontaneously change their tuning properties across days. Rats were surgically implanted with a chronic microwire assembly into the NTS or PbN. Following recovery, water-deprived rats had free access to a lick spout that delivered taste stimuli while cellular activity was recorded. In 12 rats for the NTS and 8 rats for the PbN, single units could be isolated at the same electrode on consecutive days (NTS, 14 units for 2–5 consecutive days, median = 2 days; PbN, 23 units for 2–7 days, median = 2.5 days). Waveforms were highly similar (waveform template correlation > 0.99) across days in 13 units in NTS and 13 units in PbN. This degree of similarity was rare (0.3% of pairs in NTS, 1.5% of pairs in PbN) when the waveforms were from presumed-different neurons (units recorded on nonconsecutive days with at least one intervening day in which there were no spikes, or from different wires or rats). Analyses of multi-day recordings that met this criterion for “same unit” showed that responses to taste stimuli appeared, disappeared, or shifted in magnitude across days, resulting in changes in tuning. These data imply, generally, that frameworks for cell classification and, specifically, that theories of taste coding, need to consider plasticity of response profiles.</p></div

Taste response magnitudes in spikes per sec (sps) in the NTS for taste stimuli of multiday recordings that met the “same unit” criterion.

<p>Taste response magnitudes in spikes per sec (sps) in the NTS for taste stimuli of multiday recordings that met the “same unit” criterion.</p

Correlation coefficients and Fisher z-transforms for all pairs of waveforms in the NTS and PBN.

<p>Large values indicate greater similarity of waveforms. Left panels in Fig 2a. for NTS and 2b. for PbN show the distributions of correlations for all pairs of waveforms. Right panels for Figures 2a. For NTS and 2b. for PbN show the same distributions but the proportion of the total in each category (Consecutive and Interrupted) is calculated for each group separately. The Consecutive group (black) consists of pairs of waveforms from units recorded from the same electrode across sequential days. The Interrupted group (presumed-different neurons; gray) consists of pairs of waveforms from units recorded on non-consecutive days at the same electrode, with at least one day of no activity intervening, or from different electrodes (including different animals), but only from animals that had multi-day recordings. The dashed vertical line in the right hand panels indicates the +2.58SD limit for z-transform values (for NTS, z = 3.04; for PbN, z = 2.47). The correspondence of z-transformed values to correlation coefficients is shown along the top of each plot on the right.</p

Example of a multiday recording of taste responses from a single PbN unit (#3 from Table 2).

<p>a) Rasters (top of each panel) and peristimulus-time histograms (bottom of each panel) evoked by each tastant across days. Colored triangles indicate the occurrence of a lick. b) Mean response magnitudes ± standard error of the mean (SEM) for each taste stimulus on each day. Abbreviation: CA, citric acid. This neuron showed a response to MSG that decreased across days, response to NaCl primarily on Day 2, a response to water that significantly decreased on the third day, and small responses to quinine and sucrose. c) Mean waveforms (800 μs) recorded on the three days; inset: waveform correlation between days. d) Principal components analyses results for individual waveforms recorded on each of three days, color-coded by day.</p

Pre-stimulus baseline firing rates in spikes per sec (sps) in the NTS and PbN for taste stimuli of multiday recordings that met the “same unit” criterion.

<p>Pre-stimulus baseline firing rates in spikes per sec (sps) in the NTS and PbN for taste stimuli of multiday recordings that met the “same unit” criterion.</p