The Effects of Molecular Noise and Size Control on Variability in the Budding Yeast Cell Cycle  Talia et al, Nature, 23 August 2007  William Morejón  Kelly Drinkwater

Recall the Yeast Cell Cycle  Yeast cells will grow like any other cell during the cell cycle  Undergo Budding  Results in a Mother and a Daughter  This paper is solely concerned with factors governing G1 transition to S phase in Saccharomyces Cerevisiae G1G1 D M

Variation in Length of G1  Size-dependent regulation  Cells have differential birth size (M birth )  Cells will exit G1 once they have reached a certain size (M bud )  Size-independent, fixed-time-length regulation  Molecular noise  Experimental Goal:  How much of G1 Variability is due to noise? G1 Phase

Measuring the Size Factor  Protein Based Marker for cell Size  DsRed Red Fluorescent Protein under constitutive promoter (ACT1 from actin gene)  Total Red Fluorescence per cell reflects total cell protein content  Found Exponential Growth  For small M birth, T G1 is longer  For large M birth, T G1 is smaller*Implies size factor construct M bud =M birth e αT(G1) α=growth rate

Measuring the Time Factor  If cycle timing controlled by noisy gene expression, then N x ploidy reduces variability  Used haploid, diploid and tetraploid (next slide)  Strains carry Myo1-GFP  Forms a new bud neck  Disappears at Cytokinesis*Allows us to measure G1 time

Ploidy and Noise in G1  Increased Ploidy reduces noise  WT Haploid-some noise  WT Diploid-less noise  WT Tetraploids least noise  Variability decreased by sqrt(2) for each increase in ploidy  Noise may be due to small variability in numbers of regulator molecules

Size-dependent timing in smaller cells  Relate birth size to G1 duration  Small daughter cells show strong dependency; others weak

Whi5 acts as a gatekeeper  In the nucleus, Whi5 inhibits DNA replication & budding proteins  Cyclins drive it to the cytoplasm, releasing inhibition  Divide G1 into parts T1 and T2

Size control occurs in T1  T1 varies with cell size just like overall G1 (two-slope model); very short in mothers  T2 is independent of cell size, same in mothers & daughters

Two-step model

Contributions  Separated variation in G1 length due to cell size, fixed- duration steps, and molecular noise  Demonstrated role of molecular noise due to 1/sqrt(2) change with ploidy doubling  Proposed two-step model of yeast G1 phase, with size- and time-regulated steps

What Bioengineers Can Use  Use ploidy or copy number as a noise control mechanism in engineered networks  Better control of replication in engineered yeast populations  Better understanding of replication possibly applicable to other organisms

Methods: Time Lapse Microscopy  Cells were imaged every 3 min on a fluorescent microscope  Leica DMIRE2 inverted microscope with a Ludl motorized XY stage  Budding was scored visually by the appearance of Myo1–GFP at the incipient bud neck, and division by its disappearance, generally with single-frame accuracy.  Time-lapse microscopy data were analysed with custom software written in MATLAB