Biology subjects

Crane, M. M.

Publications and source records attributed to Crane, M. M..

3 recordsLinked to original sources

bioRxiv2018

In vivo measurements reveal a single 5’ intron is sufficient to increase protein expression level in C. elegans

Introns can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). To date, the magnitude of IME has been quantified in human cell culture and plant models by comparing intronless reporter gene expression levels to those of intron-bearing reporter genes in vitro (mRNA, Western Blots, protein activity), using genome editing technologies that lacked full control of locus and copy number. Here, for the first time, we quantified IME in vivo, in terms of protein expression levels, using fluorescent reporter proteins expressed from a single, defined locus in Caenorhabditis elegans. To quantify the magnitude of IME, we developed a microfluidic chip-based workflow to mount and image individual animals, including software for operation and image processing. We used this workflow to systematically test the effects of position, number and sequence of introns on two different proteins, mCherry and mEGFP, driven by two different promoters, vit-2 and hsp-90. We found the three canonical synthetic introns commonly used in C. elegans transgenes increased mCherry protein concentration by approximately 50%. The naturally-occurring introns found in hsp-90 also increased mCherry expression level by about 50%. Furthermore, and consistent with prior results examining mRNA levels, protein activity or phenotypic rescue, we found that a single, natural or synthetic, 5 intron was sufficient for the full IME effect while a 3 intron was not. IME was also affected by protein coding sequence (50% for mCherry and 80% for mEGFP) but not strongly affected by promoter 46% for hsp-90 and 54% for the stronger vit-2. Our results show that IME of protein expression in C. elegans is affected by intron position and contextual coding sequence surrounding the introns, but not greatly by promoter strength. Our combined controlled transgenesis and microfluidic screening approach should facilitate screens for factors affecting IME and other intron-dependent processes.

genetics↗

bioRxiv2018

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC function is compromised in ageing mitotic cells. By imaging of single yeast cells during ageing, we show that the abundance of several NPC components and NPC assembly factors decreases while signs of misassembled NPCs appear. Consequently, nuclear permeability decreases, resulting in decreased dynamics of transcription factor shuttling and increased nuclear compartmentalisation. In support that declining NPC quality control is important in mitotic ageing, we find that the transport kinetics observed in ageing is mimicked in an NPC assembly mutant. Additionally, the single cell life histories reveal that cells that better maintain NPC function are longer lived. We conclude that assembly and quality control of NPCs are major challenges for ageing mitotic cells.

cell biology↗

bioRxiv2018

Dynamics of age-related catastrophic mitotic failures and recovery in yeast

Genome instability is a hallmark of aging and contributes to age-related disorders such as progeria, cancer, and Alzheimers disease. In particular, nuclear quality control mechanisms and cell cycle checkpoints have generally been studied in young cells and animals where they function optimally, and where genomic instability is low. Here, we use single cell imaging to study the consequences of increased genomic instability during aging, and identify striking age-associated genome missegregation events. During these events the majority of mother cell chromatin, and often both spindle poles, are mistakenly sent to the daughter cell. This breakdown in mitotic fidelity is accompanied by a transient cell cycle arrest that can persist for many hours, as cells engage a retrograde transport mechanism to return chromosomes to the mother cell. The repetitive ribosomal DNA (rDNA) has been previously identified as being highly vulnerable to age-related replication stress and genomic instability, and we present several lines of evidence supporting a model whereby expansion of rDNA during aging results in nucleolar breakdown and competition for limited nucleosomes, thereby increasing risk of catastrophic genome missegregation.

molecular biology↗