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Based on everyone's very helpful input on the last draft, here is a revised
draft outline for the next methods volume.  Suggestions for improvement are
welcome to [log in to unmask] and [log in to unmask]
Suggestions for authors are also welcome, although thankfully some people
have already volunteered for specific chapters.

Tentative table of contents 8/12/10   *VERY tentative thoughts about the
content of the chapters, for judging the scope overall*

Methods in Cell Biology: Tetrahymena thermophila

Introduction

1. Tetrahymena as a model organism

Systems perspectives (What has been/can be learned using Tetrahymena and how?)

2. Natural world context: what we need to know for evolutionary/phylogenetic
context, ecology, population diversity and isolation, estimates of sexual
versus asexual reproduction in the wild, seasonal behavior in native
environments? karyorelicts?

3. Nuclear dualism: MAC and MIC genomes, chromosome structures, relation of
MAC to MIC, phenotypic assortment and MAC copy number control in asexual
reproduction, nuclear differentiation in sexual reproduction, classes of IES

4. Ultrastructure: the cell overall; cortical patterning, the oral cavity,
cell pairing during conjugation

5. Membranes: cell membranes and membrane compartments; lipid profiles;
pathways for nutrient ingestion, excretion, secretion, phagocytosis,
autophagy?; preparations/purifications including rafts? Will explore models
for the evolution of mechanisms involved in membrane traffic.

6. Microtubules: overall types of structures; cilia including de/reciliation
and ciliary beating; cytoskeleton preparations/purifications including
tubulin prep

7. Chromatin: biochemical and functional characterization of histone
variants and modifications; chromatin compositions; DNA elimination;
epigenetics?; methods for ChIP

8. Genome-wide analyses: details of what genome sequencing revealed about
gene content and gene families; codon use; EST sequencing and expression
arrays; sRNA sequencing; proteomics including inventories from
organelles/secretion; resources including TGD and TGED and how they will
help to interrogate pathways

Operating principles (What approaches, tools, methods are available?)

9. Cell culture: nutrient requirements, media, temperature ranges for growth
and viability, density dependence, short term and long term working cultures
and why length of culture matters, strain storage, the stock center; growth,
stationary phase, starvation (+/- conjugation competence), conjugation;
growth without phagocytosis and/or ciliary motility

10. Strains: from T. pyriformis to T. thermophila {inbred strains and their
origin; how different are inbred strains here or below in genetics?},
collecting natural populations and their classification, the amicronucleate
condition

11. Genetics: crosses including to star (note the difference in genetic
versus physical amicronucleate condition; ie some star strains have MIC
content); genetic versus physical maps; {details of mating assays here or
below in behavior?} {strain inbreeding here or above?}

12. Transformation methods. DNA and RNA based transformation and
microinjection for the MAC and MIC; operational definitions of an essential
gene including somatic gene knockout versus knockdown; endogenous locus
tagging, transgene strategies with promoter choices; selectable markers;
antisense ribosomes, RNA silencing

13. Biochemical methods. whole cell DNA and RNA and protein; subcellular
fractionation including differential nuclei (alternatives to gum arabic),
nucleoli; principles for extracts cytoplasmic vs. nuclear vs. whole cell for
native/denaturing affinity purification; buffers, detergents, protease
inhibitors; protein/RNA/DNA tags for mass spec; MTT driven transgenes how
much Cd/Cu when, how long, how repeatedly

14. Cytological analysis: Assays of cellular ultrastructure by microscopy
(phase, IF, EM, FISH, etc., for tagged and untagged components); cell cycle
timings of replication/partition for MIC, MAC, organelles, and cell
membrane; phenotypes of perturbed cell cycle (define monsters), assays for
DNA content versus nuclear size; cell cycle mutants and what they tell us;
conjugation mutant phenotypes

15. Behavioral assays: motility (I antigens?); electrophysiology; social
interactions {mating assays here?  include costimulation, tip
transformation, homotypic vs heterotypic pairing}; mucus discharge

16. Tetrahymena in the classroom

Concluding remarks

17. Opportunities for the future

**********************************
The goal is to not have to do this again anytime soon - in other words, to
put together a volume that is more at a level of operational principles than
reviews of the current state of a field or detailed methods, which would be
available elsewhere perhaps at TGD or the stock center websites. The idea
would be to have something that one would want to read most of, or could
read all of, as an education on what has been done with Tetrahymena, what
could be done, and why the system is (or could be) particularly interesting
or enabling.