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

G1D

M

Variation in Length of G1

Size-dependent regulation Cells have differential birth size (Mbirth) Cells will exit G1 once they have reached a certain size

(Mbud)

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 Mbirth, TG1 is longer For large Mbirth, TG1 is smaller *Implies size factor

constructMbud=MbirtheαT(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 ring @ 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