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Department of Cell & Tissue Biology
513 Parnassus Avenue, HSW-615/618
San Francisco, CA 94143-0512
Microtubules are central to many processes in eukaryotic cells including chromosome
segregation, intracellular transport, and control of intracellular signaling during cell
shape remodeling. Microtubules are dynamic filaments that switch between phases
of growth and shortening, and a diverse group of proteins, called +TIPs, associates
specifically with growing microtubule plus ends in cells. End-binding proteins (EBs)
directly recognize a structural characteristic of growing microtubule ends, and recruit
almost all other +TIPs. Most +TIPs bind to EBs through short, conserved SxIP
motifs in intrinsically disordered, positively charged protein regions. Based on these
characteristics, recent bioinformatics and biochemical screening identified >30 SxIP-
motif containing +TIPs that can interact with EBs and associate with growing
microtubule plus ends in cells. As the number of EB-recruited, SxIP-motif-containing
+TIPs continues to grow, it is becoming evident that these proteins are structurally
highly heterogeneous. This structural diversity strongly suggests that +TIP
functions are also highly diverse, which raises several fundamental questions:
What are molecular functions of +TIPs? How is EB- and microtubule plus-end-association important for these functions? How are +TIPs spatially and temporally
controlled such that different +TIP complexes mediate specific microtubule
activities?
Answering these incompletely resolved questions is critical to
understanding how microtubules and +TIPs participate in normal and pathological
cell behaviors, and is one of our long-term goals.

The unexpected diversity of SxIP-motif-containing proteins also challenges earlier
views that +TIPs mainly regulate MT polymerization dynamics. Instead, a central
hypothesis of our research is that a subgroup of +TIPs function as adaptors that
promote spatially and temporally controlled capture of EB-positive growing
microtubule ends to polarize microtubule-dependent activities
and that microtubule
plus-end-tracking observed in cells may be a byproduct rather than the primary
function of these proteins. Such search-and-capture of intracellular structures by
growing microtubules was initially proposed as a mechanism by which dynamic
microtubules are selectively stabilized at the cell cortex or at kinetochores during
mitosis. However, search-and-capture in its simplest form in which growing
microtubules randomly hit intracellular targets is highly inefficient on a cell
biological timescale, and +TIPs may function as facilitators of microtubule capture
and stabilization. Such +TIPs would be targeted through binding domains that
provide a receptor for EB-covered growing microtubule ends, and thus mark specific
intracellular structures as microtubule interaction sites.

One example are CLASP protein clusters that accumulate around cell-matrix
adhesions. Such focal adhesions (FAs) mediate cell interactions with the
extracellular matrix, and controlled FA turnover is fundamentally important for
directed cell migration. Abnormal cell-matrix interactions contribute to cancer
metastasis and malignancy, and have also been implicated in fibrosis and aberrant
wound healing in autoimmune diseases. MTs play a role in regulating FA dynamics,
but the underlying molecular mechanisms are not understood. We propose that
+TIP-mediated FA-associated matrix remodeling is central to the initiation of FA
turnover, and we are investigating underlying molecular mechanisms. Since matrix
remodeling is central to metastatic tissue remodeling and cancer cell invasion,
which accounts for the majority of cancer-related deaths, elucidating the molecular
details of this pathway has important clinical implications. EB1 was originally
identified as a binding partner of the adenomatous polyposis coli (APC) tumor
suppressor protein that is mutated in >90% of colorectal cancer cases. Recent
proteomics studies further found that EB1 is overexpressed in colorectal cancers
with poor prognosis, and in liver and breast cancer. A function of EB-dependent
+TIP interactions during cancer cell invasion is also indicated by our recent work,
using 3D tissue culture of epithelial remodeling to demonstrate that EB1 is required
for coordinated cell migration into a collagen matrix. Thus, in addition to
establishing new paradigms relating to +TIP functions, our research is highly
relevant to understanding pathological cell behavior.





EB1 +TIP interaction modes
mCherry-tubulin and EB1-EGFP
(Spinning disk confocal, 7.5x real time)
Focal adhesion turnover
during 3D epithelial remodelling
Paxillin-mCherry and EGFP-CLASP2
during epithelial sheet migration,
~1000x real time