Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

Chlamydomonas reinhardtii IFT particle trains, important for flagella maintenance and assembly, are observed to decrease in size as a function of cilia length

Flagellar length control in Chlamydomonas reinhardtii, experimental and mathematical approaches

Cells maintain organelles at precise size and orientation without a ruler or compass. In this dissertation I examine how these unintelligent, complex mixtures of molecules are able to correctly maintain the length of their cilia [or flagella] over a range of perturbing conditions. In other words, how do cells measure?I treat the problem at three different spatial scales using the flagella of the proven model organism for mammalian cilia, Chlamydomonas reinhardtii. Chlamydomonas cells possess a pair of ~12 micron flagella that they use for motility. The flagella are built and maintained from their distal tips by the active movement of precursor proteins to and from the cell body in the process known as intraflagellar transport (IFT). Using TIRF microscopy and computational methods, developed in Chapter 2, I found that the rate of IFT (amount of protein/second) sets the growth rate of the flagellum, as evidenced by higher IFT rate in lengthening flagella and lowered IFT rate in shortening flagella. Furthermore, the IFT rate is set by the amount of IFT material localized at the flagellar base in a length-dependent manner. Surprisingly, I found that the dynamics of IFT particle entry into the flagellum are consistent with the mathematics of an avalanching system (Chapter 3). From a whole cell systems perspective, it has been unclear whether cells have separate programs to directly shorten and lengthen their flagella or whether shortening and lengthening are part of an overall size control program. The main evidence for separate programs comes from experimental amputations of single flagella in normal biflagellate cells and in cells that abnormally possess extra flagella. I repeated these classical experiments in more controlled conditions using microfluidic cell trapping and laser amputation of flagella (Chapter 4). I found by mathematical modeling that an overall size control program fits the data exceptionally well. The results indicate that flagella self-assemble but ultimately size is controlled through the cytoplasmic availability of precursor proteins. This finding provides a general model for size control in dynamic organelles. Using the mathematical model, I developed an experimental protocol to compute the turnover rate of proteins incorporated into the axoneme, the structural part of the flagellum (Chapter 5). Finally, I derive quantitative versions of flagellar length control models and find evidence for a volumetric diffusion model, whereby organelle volume is sensed through a Ran-GTP gradient, such as is used to regulate nuclear import. This general model could apply to linear organelles like flagella as well as to spherical ones like the nucleus or non-uniformly shaped organelles such as the endoplasmic reticulum (Chapter 6)

Development Data

Raw development time data for Drosophila melanogaster raised with all possible combinations of the five gut bacterial species examined in this study

Fecundity Data for Antibiotic Treatments

Fecundity data for Drosophila melanogaster under different experimental conditions and fed various combinations of five species of gut bacteria

Survival Data

Raw survival data for Drosophila melanogaster raised with all possible combinations of the five gut bacterial species examined in this study

Development Time Data Under Different Conditions

Development time data for Drosophila melanogaster under different experimental conditions and fed various combinations of five species of gut bacteria

Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model.

The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly