Cellular morphology is established and maintained through the dynamic assembly and regulatory processes of molecular components,
which provide a variety of spatial structures to express a given cellular function. Cells sense their surrounding environment
via mechanical and chemical stimuli, such as forces, cell-cell and cell-extracellular matrix (ECM) interactions or gradients
of signaling molecules, and they respond by developing an axis of polarity. The key element controlling cell shape is the cytoskeleton.
The cytoskeleton consists of distinct filamentous systems: actin filaments (thin filaments), intermediate filaments, microtubules,
and an emerging non-canonical cytoskeleton septin. These are all polymers, having the ability to reversibly assemble and disassemble.
Intermediate filaments and septins confer cortical rigidity and actin filaments generate the driving force for cell migration and the
form to provide cell shape, while microtubules serve as tracks for the directional transport of molecular components or drag ropes for the movement of organelles.
Microtubules for intracellular traffic
The microtubule has an intrinsic structural polarity, which is fundamental to the directional transport mediated by motor proteins
(Vallee RB and Sheetz MP, Science. 1996, 271, 1539-1544.; Hirokawa N, Science. 1998, 279, 519-526.).
The appropriate delivery of physiologically active substances is crucial for their asymmetric distribution;
therefore, the control of directionality and organization of microtubules in cells is essential to cellular morphogenesis and functioning.
Thus, far from being mere structural elements, microtubules are key determinants of cell polarity, and thus microtubule dynamics are a major target of signaling pathways.
Microtubules are self-organized by the polymerization of alpha-/beta-tubulin heterodimers that are arranged parallel to a cylindrical axis,
with alpha-tubulin and beta-tubulin having exposed minus and plus ends, respectively. In a population of microtubules in a steady state of assembly/disassembly,
an individual microtubule interconverts stochastically between periods of slow growth and rapid shortening at the plus end,
a behavior known as "dynamic instability" (Mitchison T and Kirschner M, Nature. 1984, 312, 237- 42.; Horio T and Hotani H, Nature. 1986, 321, 605-7.).
Minus ends are generally not dynamic and rapidly depolymerize if these are not capped and stabilized.
In cells, dynamic instability has a functional role in controlling the disposition of the microtubule arrays in response to external stimuli.
The fast disassembly provides a means for the rapid re-organization of the microtubules in response to changing cellular requirements.
Microtubule organization in cells
If one looks at the pattern of microtubules in a single cultured cell that is not in contact with surrounding cells,
one will generally see microtubules distributed with their minus ends clustered together around the cell center and their plus ends extending out into the cytoplasm.
This radial plus end-out orientation of microtubules is established through the combination of microtubule-dependent transport of minus end-anchoring materials
by a minus end directed molecular motor, typified by dynein, and growth of the plus ends.
A cell is polarized when it has developed a main axis of organization following a trigger by external signals.
Accompanying cell polarization, the microtubule network can be regulated by numerous tubulin/microtubule modulators.
The basic concept explaining the generation of microtubule asymmetry, termed the "selective stabilization model" or "search-and-capture model",
involves local stabilization of a subset of microtubules (Kirschner M and Mitchison T, Cell. 1986, 45, 329-42).
In this model, dynamic instability allows microtubules to search stochastically the three-dimensional space within cells
in order to find and capture specific target sites on the cell periphery that have been activated by environmental signals .
The plus ends attaching to the cell cortex are stabilized at their ends by local factors, resulting in increased lifespan of the entire microtubule.
Different microtubule dynamics in a resting cell and a cell undergoing active migration
Fluorescent protein: beta-tubulin-GFP; Cell: Xenopus A6; Playback speed: 12 frame/sec.
I collected these movies around 1998.
Seeing this I believed that yet unidentified factors must be present at the individual microtubule ends to control their behavior, and started to search for the factor.
In our efforts to clarify the molecular mechanisms underlying the microtubule patterning, we discovered an intriguing class of microtubule-associated proteins (MAPs),
"plus-end tracking proteins (+TIPs)", which are accumulating at the microtubule ends
(Mimori-Kiyosue Y et al., J Cell Biol. 2000, 148, 505-18; Mimori-Kiyosue Y et al., Curr Biol. 2000, 10, 865-8; Schuyler SC and Pellman D, Cell, 2001, 105, 421-4).
Microtubule plus-end-tracking proteins (+TIPs)
To date, it has become clear that +TIPs are involved in the microtubule organization (Mimori-Kiyosue Y and Tsukita S, J Biochem(Tokyo) 2003, 134, 321-6).
+TIPs are a diverse group of specialized MAPs that are evolutionarily conserved and that accumulate at the ends of growing microtubules
(Akhmanova A and Steinmetz MO, Nat Rev Mol Cell Biol, 2008, 9, 309-22).
Many +TIPs are targeted to the growing plus ends through interaction with EB1 (end-binding 1) family proteins (EB1, EB2, EB3).
EBs are autonomous plus end-binding proteins recognizing the end-specific tubulin structure independently of any binding partners
(Mimori-Kiyosue Y et al., Curr Biol. 2000, 10, 865-8; Bieling, P et al., Nature, 2007, 450, 1100-5).
Thus, EB1 family proteins are core components of +TIP complexes.
Some +TIPs, such as CLIP-associating proteins (CLASPs), actin crosslinking family 7 (ACF7) and adenomatous polyposis coli (APC) tumor suppressor protein,
localize to the cell cortex near migrating cell edges and attach EB1-positive microtubule plus ends to the cortex and facilitate directional migration
(Mimori-Kiyosue Y et al., J Cell Biol. 2000, 148, 505-18; Mimori-Kiyosue Y et al., J Cell Biol. 2005, 168, 141-53).
These factors exert microtubule stabilizing or growth promoting effects, and thus polarize microtubules toward the leading edges of the cell.
In polarized epithelial cells, in which microtubules are aligned along the apicobasal polarity with their plus ends facing toward the basal side,
CLASPs and APC are localized at the basal cortex and retain microtubule density at these structures (Hotta A et al., J Cell Biol, 2010, 189, 901-17).
Until today, it has been almost established that +TIPs are crucially involved in the generation of polarized microtubule arrays.
Green: EB1-GFP; Red: TagRFP-tubulin; Cell: MEF; Playback speed: 12 frame/sec
*Original movies with higher resolution are available upon request.
Cortical +TIPs-anchoring factors
Above class of +TIPs are attached to the inner surface of cells by cortical proteins.
For example, CLASPs are associated with the basal cell cortex by PH domain-containing proteins LL5alpha and LL5beta
(Lansbergen G et al., Dev Cells, 2006, 11, 21-32; Hotta A et al., J Cell Biol, 2010, 189, 901-17).
The accumulation of LL5s to the cell cortex is initiated by integrin-laminin association,
and in turn LL5s stabilize the localization of a subset of laminin receptor integrins at the basal plasma membranes.
It is possible that microtubules anchored by LL5s could affect laminin-mediated cell adhesion and migration in epithelia, as well as other types of differenciated tissue structures.
Then what is the biological importance of this highly organized microtubules?
Considering that the microtubules are the rails for the directional intracellular trafficking,
the microtubule-attachment sites must be serving as the terminal for loading and unloading of cargos.
So far, however, no specific cargos that are delivered to these sites have been identified.
Thus, our current focus is to identification and analysis of the cargos that are delivered to the microtubule-attachment sites and secreted to the outside of cells,
and then control the function of surrounding cells in tissues.
"Three-dimensional tracking of plus-tips by lattice light-sheet microscopy permits the quantification of microtubule growth trajectories within the mitotic apparatus"
Norio Yamashita, Masahiko Morita, Wesley R. Legant, Bi-Chang Chen, Eric Betzig, Hideo Yokota, Yuko Mimori-Kiyosue.
J. Biomed. Opt. 20(10), 101206. doi:10.1117/1.JBO.20.10.101206
[PDF]Supplementary materials for our JBO paper
This material includes a detailed description how we optimized Imaris settings for EB1-GFP tracking to analyze the lattice light-sheet microscope data and movie legends.