Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore–microtubule attachment, chromosome alignment‐maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc‐finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1‐dependent phosphorylation at multiple sites during mitosis. CAMP‐depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K‐fibres to the tension exerted upon establishment of sister kinetochore bi‐orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C‐terminal region containing the zinc‐finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP‐E and CENP‐F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP‐E and CENP‐F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore–microtubule attachment during bi‐orientation.

Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.

In the process of cancer spreading, different modes of invasion exist. One is expansive invasion, in which a group of cancer cells gradually expands along with cancer cell proliferation. Invasion of cancer cells is also modified by their interaction with stromal cells including cancer-associated fibroblasts (CAFs). Cancer cells co-invade with CAFs, and invasion by CAFs frequently precede invasion by cancer cells, which indicates CAF-led cancer cell invasion. Here, we show that CAFs induce apoptosis in gastric cancer cells, which prevented expansive invasion by cancer cells and instead facilitated CAF-led invasion. Death receptor 4 and activation of caspase-8 in cancer cells mediated cancer cell apoptosis induced by CAFs, which was dependent on contact between cancer cells and CAFs. Apoptotic cancer cells in turn released apoptotic vesicles and stimulated invasion of CAFs. Accordingly, cancer cells followed the migrating CAFs. Treatment with a caspase inhibitor, ZVAD, or forced expression of a death domain fragment in cancer cells prevented cancer cell apoptosis induced by CAFs and increased expansive invasion by cancer cells in extracellular gel invasion assays, while the rate of cancer cell invasion led by CAFs was decreased. Death domain-fragment expression also prevented intramural invasion by gastric cancer cells in the stomach. Because CAF-led invasion is characterized by the movement of individual cancer cells away from the tumour, adequate cancer cell apoptosis may promote cancer dissemination.

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Most cancer cells are aneuploid, which could be caused by defects in chromosome segregation machinery. Nucleoporins (Nup) are components of the nuclear pore complex, which is essential for nuclear transport during interphase, but several nucleoporins are also known to be involved in chromosome segregation. Here we report a novel function of Nup188, one of the nucleoporins regulating chromosome segregation. Nup188 localizes to spindle poles during mitosis, through the C‐terminal region of Nup188. In Nup188‐depleted mitotic cells, chromosomes fail to align to the metaphase plate, which causes mitotic arrest due to the spindle assembly checkpoint. Both the middle and the C‐terminal regions were required for chromosome alignment. Robust K‐fibers, microtubule bundles attaching to kinetochores, were hardly formed in Nup188‐depleted cells. Significantly, we found that Nup188 interacts with NuMA, which plays an instrumental role in focusing microtubules at centrosomes, and NuMA localization to spindle poles is perturbed in Nup188‐depleted cells. These data suggest that Nup188 promotes chromosome alignment through K‐fiber formation and recruitment of NuMA to spindle poles.