Jennifer S. Tirnauer

Determining the position of the cell division plane

Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells—a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.

THE APC-ASSOCIATED PROTEIN EB1 ASSOCIATES WITH COMPONENTS OF THE DYNACTIN COMPLEX AND CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

Mitotic spindle misorientation in cancer--out of alignment and into the fire.

Mitotic spindle orientation can influence tissue organization and vice versa. Cells orient their spindles by rotating them parallel or perpendicular to the cell – and hence the tissue – axis. Spindle orientation in turn controls the placement of daughter cells within a tissue, influencing tissue morphology. Recent findings implicating tumor suppressor proteins in spindle orientation bring to the forefront a connection between spindle misorientation and cancer. In this Commentary, we focus on the role of three major human tumor suppressors – adenomatous polyposis coli (APC), E-cadherin and von Hippel-Lindau (VHL) – in spindle orientation. We discuss how, in addition to their better-known functions, these proteins affect microtubule stability and cell polarity, and how their loss of function causes spindles to become misoriented. We also consider how other cancer-associated features, such as oncogene mutations, centrosome amplification and the tumor microenvironment, might influence spindle orientation. Finally, we speculate on the role of spindle misorientation in cancer development and progression. We conclude that spindle misorientation alone is unlikely to be tumorigenic, but it has the potential to synergize with cancer-associated changes to facilitate genomic instability, tissue disorganization, metastasis and expansion of cancer stem cell compartments.

Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine.

A major feature of epithelial cell polarity is regulated positioning of the mitotic spindle within the cell. Spindles in cells of symmetrically expanding tissues are predicted to align parallel to the tissue plane. Direct measurement of this alignment has been difficult in mammalian tissues. Here, we analyzed the position of spindles in intact mouse intestinal epithelium using microtubule immunofluorescence and three-dimensional confocal imaging. Mitotic cells were identified in the proliferative zone of intestinal crypts. Spindle angle relative to the apical cell surface was determined either by direct measurement from confocal images or with a computational algorithm. Angles averaged within 10° of parallel to the apical surface in metaphase and anaphase cells, consistent with robust planar spindle positioning, whereas spindles in prometaphase cells showed much greater angle variability. Interestingly, cytokinetic furrows appeared to extend from the basal cell surface toward the apical surface. This type of image analysis may be useful for studying the regulation of spindle position during tissue remodeling and tumor formation.

Spindle misorientation in tumors from APCmin/+ mice

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in the majority of colon cancers, and its mutation may initiate cancer by multiple mechanisms. Recently, abnormal mitotic spindle orientation was shown in normal‐appearing tissues from mice heterozygous for APC mutation. To determine the effect of APC mutation on spindle orientation in tumors, and to better understand its mechanism, we measured mitotic spindle orientation in intestinal tumors from APC mutant mice, with three‐dimensionally reconstructed confocal stacks of microtubule immunofluorescence images. We found spindle angles were increased in crypts heterozygous for the APCmin mutation, and further increased in tumors. Astral microtubules of these spindles were clearly evident, suggesting astral microtubule loss is not a major mechanism of spindle misorientation in intestinal cells lacking wild‐type APC. β‐Catenin staining was markedly abnormal in crypts and tumors from the mutant mice, suggesting a possible role in spindle orientation. Spindle angles in colon tumors with wild‐type APC were equivalent to those in wild‐type mice, showing that spindle misorientation is specific to APC mutation and not a general feature of tumors. We suggest spindle misorientation may contribute to the loss of normal tissue organization during tumor formation and could offer new insights into early carcinogenic events.

CK1 activates minus-end–directed transport of membrane organelles along microtubules

This study shows that the signal transduction pathway responsible for the initiation of minus-end–directed movement of membrane-bounded pigment granules in melanophores involves sequential activation of protein phosphatase 2A and casein kinase 1 and that this activation correlates with increased phosphorylation of the dynein intermediate chain.

The LKB1 tumor suppressor controls spindle orientation and localization of activated AMPK in mitotic epithelial cells.

Orientation of mitotic spindles plays an integral role in determining the relative positions of daughter cells in a tissue. LKB1 is a tumor suppressor that controls cell polarity, metabolism, and microtubule stability. Here, we show that germline LKB1 mutation in mice impairs spindle orientation in cells of the upper gastrointestinal tract and causes dramatic mislocalization of the LKB1 substrate AMPK in mitotic cells. RNAi of LKB1 causes spindle misorientation in three-dimensional MDCK cell cysts. Maintaining proper spindle orientation, possibly mediated by effects on the downstream kinase AMPK, could be an important tumor suppressor function of LKB1.

LKB1 Destabilizes Microtubules in Myoblasts and Contributes to Myoblast Differentiation

Background Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. Findings We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMP-activated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. Conclusions Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeletal changes required for differentiation. Transient destabilization of microtubules might be a useful strategy for enhancing and/or synchronizing myoblast differentiation.

Bone matrix osteonectin limits prostate cancer cell growth and survival.

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on osteonectin-null matrix. Our data support the idea that osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.

Spontaneous spheroid budding from monolayers: a potential contribution to ovarian cancer dissemination

Summary Ovarian cancer is the most lethal gynaecologic cancer, in large part because of its early dissemination and rapid development of chemotherapy resistance. Spheroids are clusters of tumor cells found in the peritoneal fluid of patients that are thought to promote this dissemination. Current models suggest that spheroids form by aggregation of single tumor cells shed from the primary tumor. Here, we demonstrate that spheroids can also form by budding directly as adherent clusters from a monolayer. Formation of budded spheroids correlated with expression of vimentin and lack of cortical E-cadherin. We also found that compared to cells grown in monolayers, cells grown as spheroids acquired progressive resistance to the chemotherapy drugs Paclitaxel and Cisplatin. This resistance could be completely reversed by dissociating the spheroids. Our observations highlight a previously unappreciated mode of spheroid formation that might have implications for tumor dissemination and chemotherapy resistance in patients, and suggest that this resistance might be reversed by spheroid dissociation.