Page 166 - PC2019 Program & Proceedings

PLANT CANADA 2019

S117. Identification and characterization of novel targets for a subfamily of Arabidopsis
calmodulin-like (CML) proteins
*
Teresinski, H. ; W. Snedden
Queen's University

Calcium ions serve as ubiquitous second messengers in eukaryotes. Calcium sensor proteins, such as
calmodulin (CaM), detect and transduce calcium signals by regulating downstream target proteins. In
plants, the largest family of calcium sensors are the CaM-like (CML) proteins: Arabidopsis has 7 CaMs
and 50 CMLs. Some CMLs are known to function in development and stress-response but most remain
unstudied. Whereas many downstream targets of CaM are well characterized, very few CML targets have
been identified. Here, using a range of biochemical and molecular approaches, we have identified several
target proteins that interact exclusively with a small subfamily of Arabidopsis CML paralogs. Our data
indicate that these particular CMLs display unusual calcium-binding and structural properties relative to
other members of the CML family. Using the yeast two-hybrid system, we identified putative targets of
these CMLs, delineated the CML-target interaction domains, and corroborated the high specificity of this
interaction using in vitro assays and the in planta split-ubiquitin system with Nicotiana
benthamiana. Structural and biophysical analyses indicate the presence of both CaM and CML binding
sites on the target proteins that we have identified. We will discuss proposed roles for these CMLs and
their targets in cytoskeletal function based on our analyses using T-DNA insertion knockout-lines,
subcellular localization analyses using GFP-CML fusion proteins, and promoter activity assays using
CML promoter:GUS reporter transgenic plants.

Howard Teresinski (h.teresinski@queensu.ca)

S118. Make or break? Microtubule growth and shrinkage are controlled by dynamic turnover of
plus-end proteins
2
*1
1
Halat, L. ; R. Eng ; D. Coombs ; G. Wasteneys
1
1 University of British Columbia
2 Max Planck Institute of Molecular Plant Physiology
Assembly and disassembly of microtubule (MT) polymers is critical for plant development and response
to environmental signals. In Arabidopsis thaliana, MICROTUBULE ORGANIZATION 1 (MOR1) acts
as a MT polymerase by rapidly adding tubulin dimers to the growing (plus) ends of MTs. By contrast,
Armadillo Repeat Kinesins (ARKs) are motor proteins that act as catastrophe factors by promoting MT
disassembly. The indispensable role of MOR1 is well established, but its mechanism of polymerase
activity and association with catastrophe factors remains elusive. To investigate MOR1 kinetics in
vivo,we used homologous recombination to engineer yellow fluorescent protein (YFP) tagged wild-type
MOR1 and a mutant form of the protein (mor1-1) that has been shown to reduce MT polymerization
rates. Using Total Internal Reflection Fluorescence microscopy, we performed fluorescence recovery
after photobleaching (FRAP) on growing MTs. Compared to the wild-type MOR1-YFP, the mutant mor1-
1-YFP protein recovered fluorescence very slowly, which indicated MOR1 normally has a transient
association with the growing MT that is tightly coupled to MT growth. We next hypothesized that ARKs
promote MT disassembly by interfering with MOR1’s affinity for MTs. Using CRISPR/Cas9, we
simultaneously knocked out expression of the functionally redundant ARKs 1and 2, and assessed MOR1
dynamics by FRAP. Through these strategies and innovative techniques for live cell imaging, we have
generated a new model on the fundamental process of MT dynamics.

Laryssa Halat (laryssahalat@gmail.com)

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