The Cytoskeleton •Organizes and stabilizes cells •Pulls chromosomes apart •Drives intracellular traffic •Supports plasma membrane and nuclear envelope •Enables cellular movement •Guides growth of the plant cell wall •Provides machinery for muscle contraction and nerve cell extension •Controls the amazing diversity of eukaryotic cell shapes Three major types of cytoskeleton •Microtubules •Actin Filaments (Microfilaments) •Intermediate Filaments (still debated for plants) •Cytoskeleton can be visualizes simply by staining for protein - Coomassie blue here. All filaments are assembled from smaller protein sububits Sububits of filaments: Actin for Microfilaments Tubulin for Microtubules IF protein for IFs “Cytoskeletal systems are dynamic and adaptable, organized more like an ant trail than like interstate highways”. Microtubules - Tubulin Microfilaments -Actin Intermediate Filaments Evolutionary Conservation •Actin and tubulins are highly conserved among organisms. •Actin amino acid sequence is usually 90% identical (great control antibody!). •IF proteins less conserved. Cytoplasmic IF proteins mostly in metazoan lines, nuclear IF proteins (lamins) more widely conserved, possibly ancestral. •Actin and MTs bind to so many proteins that sequence might be very constrained. Concepts: Thermal stability and structure Structure of MT and MF allows for both stability and dynamic ends. Structure of IF more “rope-like” due to staggered long subunits. ATP binding to actin promotes its polymerization Treadmilling Concepts: Two ends of filament have different on/off rates (due to conformation of monomers. Monomer carries NTF (Actin: ATP, tubulin: GTP). Slow hydrolysis occurs in polymer. Critical concentration is different for plus and minus end: Results in net assembly at plus end and net disassembly at minus end. Treadmilling of a microtubule in vivo Microinjection of rhodamin-labeleld tubulin (1:20 with unlabeled tubulin). Dark mark from unlabeled tubulins slides less to the right than ends do. Dynamic Instability Loss of “GTP cap” at the plus end of the filament. Leads to rapid shrinkage of plus end. After free tubulin concentration increases, “rescue” occurs. Dynamic instability- subunit conformation change Dynamic instability" Dynamic instability and cellular organization Nucleation In a test-tube situation, nucleation is slow because cooperativity increases assembly rate in larger filament. polymerized fragments can act as seeds to “nucleate” filaments In the cell, nucleation is favored by nucleating proteins. Microtubule Nucleation Microtubule Organizing Center (MTOC): Site in the cell where MT nucleation starts: Centrosomes, in plants outer nuclear envelope, in yeast spindle pole bodies. Also non-mitotic MTOCs, in plants for cortical MTs. γ-Tubulin ring complex: γ-TuRC, γ- Tubulin plus additional conserved proteins, found at plant NE and cell cortex. Nucleation can be initiated on isolated plant nuclei. B shows purified γ-TuRCs and single MTs nucleating from them. Typically at the Actin Nucleation plasma membrane. Actin-related proteins (ARP2/ARP3) and additional proteins corm “Cap” at minus end of actin filament. Cap can also bind to existing filaments, thereby promoting branched network. ARPs are similar to actin at plus end, dissimilar at minus end: stops growth at minus end. Branched actin networks in vitro: mixed subunits and purified ARP complexes Arabidopsis ARP2 and ARP3:WURM and DISTORTED1 Distorted cell shapes, here trichomes and epidermal pavement cells. Distorted arrangement of actin skeleton. Plant Cell. 2003 Jul;15(7):1632-45. Filament elongation is regulated by proteins that bind to free subunits: thymosin and profilin for actin Observation: At concentration > 50 fold over critical conc. Still 50% free actin in cell Plant profilin Overexpression (B,E,H) and underexpression (C,F,I) of an Arabidopsis profilin. Phenotypes mostly based on increased and decreased cell elongation, no drastic shape changes; Involved in actin skeleton formation, but not in shape determination. (Plant Physiol. 2000 Dec;124(4):1637-47.) . Stathmin regulates MT polymerization Stathmin binding prevents addition to microtubules by binding to two dimers, reduces free concentration of dimers, (analogous to the drug colchicin). Stathmin is regulated by phosphorylation, downstream of signaling kinases e.g. Ca/calmodulin dep. Kinase (in mammals) Filament-binding proteins 1. MAPs. Microtubule-associated proteins. Can stabilize MTs and connect MTs with other MTs and with other cellular components. End-binding proteins affect filament stability MAPs like XMAP215 bind preferentially to GTP end and stabilize it. Kinesins like Kin1, KIF2 (“catastrophin”) counteract and destabilize plus end. Capping proteins can target MT ends to cellular structures. Here, EB1directs microtubule plus end to site in yeast bud. Using EBI1 to follow the MT plus ends in live cells" Actin cross-linking proteins Typically they have two actin-binding sites at a certain distance. Distance determines geometry of the cross-linked structure. Severing proteins Severing has opposing effects on filament growth above and below the critical monomer concentration. Proteins: katanin for MTs, gelsolin for actin. Katanin: 2 Sus, smaller hydrolyses ATP and severes MTs, larger directs to centrosomes. Releases MTs from MTOC. In vitro demonstration of katanin activity Top: Microtubules were stabilized on glass slide with taxol, and stained with rhodamine. When purified severin (and ATP) is added, numerous breaks are observed after 30 minutes of incubation (bottom). Fragile fiber (fra) mutant in Arabidopsis defines a plant katanin •Fra mutants have reduced mechanical strength in hypocotyl. •Fra2 encodes katanin 60. •Reduced number of cortical MT bundles (b), might weaken cell wall (fewer cellulose fibers) Fra2 phenotype and mechanical strength Morphology of the Wild Type and the fra2 Mutant. (A) Morphology of 8-week-old plants. The main inflorescence stem of a fra2 plant (right) is much shorter than that of a wild-type plant (left). (B) and (C) The main inflorescence stems. The fra2 stem (C) has reduced internode length compared with the wild-type stem (D) and (E) Siliques of wild type (D) and fra2 (E). (F) and (H) The rosette leaves of a 5-week-old fra2 plant (H) are more compact than those of a wild-type plant (F). (G) and (I) Individual leaves of 5-week-old plants. The lengths of both blades and petioles in fra2 (I) are reduced compared with those of the wild type (G). From left to right, the leaves are arranged according to the order from cotyledons to the youngest leaves. Original screen: breaking strength of hypocotyls. The main inflorescence stems of 8-week-old plants were divided into four equal segments and measured for the force required to break the stems. Segments were numbered in order from the top to the bottom of the stems. Plant Cell, Vol. 13, 807-828, April 2001 Gelsolin is an actin-severing protein Can bind at both the side and the end of the filament: After severing, gelsolin becomes cap. Increasing amounts of poppy gelsolin added to rhodamin-labeled, in vitro polymerized actin .